CDP-Diacylglycerol Synthetase Coordinates Cell Growth and Fat Storage through Phosphatidylinositol Metabolism and the Insulin Pathway

<div><p>During development, animals usually undergo a rapid growth phase followed by a homeostatic stage when growth has ceased. The increase in cell size and number during the growth phase requires a large amount of lipids; while in the static state, excess lipids are usually stored in adipose tissues in preparation for nutrient-limited conditions. How cells coordinate growth and fat storage is not fully understood. Through a genetic screen we identified <i>Drosophila melanogaster</i> CDP-diacylglycerol synthetase (CDS/CdsA), which diverts phosphatidic acid from triacylglycerol synthesis to phosphatidylinositol (PI) synthesis and coordinates cell growth and fat storage. Loss of <i>CdsA</i> function causes significant accumulation of neutral lipids in many tissues along with reduced cell/organ size. These phenotypes can be traced back to reduced PI levels and, subsequently, low insulin pathway activity. Overexpressing <i>CdsA</i> rescues the fat storage and cell growth phenotypes of insulin pathway mutants, suggesting that <i>CdsA</i> coordinates cell/tissue growth and lipid storage through the insulin pathway. We also revealed that a DAG-to-PE route mediated by the choline/ethanolamine phosphotransferase Bbc may contribute to the growth of fat cells in <i>CdsA</i> RNAi.</p></div

<i>CdsA RNAi</i> affects PI metabolism and insulin pathway activity.

<p>(A) tGPH reporter assay in salivary gland cells. The cell membrane fluorescence signal is significantly weaker and more diffuse in <i>CdsA RNAi</i> than in the <i>ppl-Gal4</i> control. Images were taken with the same exposure time. Membrane-to-cytoplasmic GFP intensity ratios were calculated from measurements of mean pixel intensities within equal areas of membrane versus cytoplasm. Histogram: n = 21. (B) Total Akt and phosphorylated Akt (Ser505) levels were detected by western blotting. α-tubulin (Tub) was used as a loading control. Average and standard deviation of relative band intensity ratio of p-Akt <i>versus</i> total Akt from three replicates is indicated at the top after normalization. The Western blot result from one experiment is shown here. In whole larva and salivary gland, RNAi of <i>CdsA</i> diminished Akt phosphorylation at serine505. (C) Phospholipid levels obtained by lipid profiling of whole larva or salivary gland samples from wandering 3^rd instar larvae with the following genotypes: <i>Gal4</i> control (<i>G4/+</i>), <i>CdsA RNAi</i>, and <i>CdsA</i> overexpression (<i>CdsA EP</i>). <i>CdsA</i> was knocked down with <i>ppl-Gal4</i> in salivary gland and <i>tub-Gal4</i> in whole larvae. Assays were done in triplicate. Note that the PI level in the <i>CdsA RNAi</i> salivary gland sample is less than 10% of that in the <i>Gal4</i> control. The levels of PI, PA, and PG are normalized to total phospholipids. (D) PIP2 and PIP3 levels measured by mass spectrometry and ELISA kit, respectively. Assays were done at least in triplicate. The level of PIP2 is normalized to total phospholipids. The level of PIP3 is normalized to dry weigh. (E) Silencing <i>Pis</i> reduces salivary gland size and causes accumulation of lipid droplets. Blue: DAPI staining for nuclei; red: Nile red staining for neutral lipids. Histogram: n = 9. (F) Phospholipid levels obtained by profiling of salivary gland samples from control and <i>Pis RNAi</i> wandering 3^rd instar larvae. Assays were done in triplicate. The levels of PI and PG are normalized to total phospholipids. (G) <i>Pis</i> mutant salivary gland cells (marked by the absence of RFP, dashed white circles) are significantly smaller than neighboring wild-type cells. <i>Pis</i> mutant salivary gland clones were induced during embryogenesis and visualized in wandering 3^rd instar larvae. Blue: DAPI staining for nuclei; green: BODIPY staining for neutral lipids; red: Ubi-mRFP, which marks cytosol. Histogram: n = 11. Scale bar (A, E, G): 50 µm. Error bars (A, C, D, E, F, G) represent SEM. (*) P<0.05; (**) P<0.01; (***) P<0.001 (Student's t-test).</p

<i>CdsA</i> broadly affects fat storage.

<p>(A) Simplified schematic of phospholipid and glycerolipid synthesis. FA-CoA: Fatty acyl CoA; LPA: lysophosphatidic acid; PA: phosphatidic acid; DAG: diacylglycerol; TAG: triacylglycerol; CDP-DAG: cytidine diphosphate diacylglycerol; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PI: phosphatidylinositol; PG: phosphatidylglycerol. CdsA adds CTP to PA and generates CDP-DAG; Pis catalyzes the donation of the phosphatidyl group from CDP-DAG to inositol and produces PI, which is the precursor of all PI derivatives, such as PIP2 and PIP3; Bbc synthesizes PC and PE from DAG, which can also be converted to TAG by DGAT. (B) (<i>left</i>) RNAi knockdown of <i>CdsA</i> in salivary gland causes massive lipid accumulation. <i>pplG4/+</i>: the <i>ppl-Gal4</i> driver only; <i>ppl>CdsA RNAi</i>: <i>UAS-CdsA RNAi</i> driven by <i>ppl-Gal4</i>. Blue: DAPI staining for nuclei; red: Nile red staining for neutral lipids. (<i>right</i>) TAG levels of fat body-removed whole larval samples measured by mass spectrometry. The level of TAG is normalized to total phospholipids. (C) <i>CdsA</i> affects fat storage in many tissues. In wandering 3^rd instar <i>tub</i>><i>CdsA RNAi</i> larvae, massive fat storage in many non-adipose tissues is detected by Nile red staining. Blue: DAPI staining for nuclei; red: Nile red staining for neutral lipids. (D) <i>CdsA RNAi</i> reduces salivary gland, brain, and wing disc tissue size. <i>CdsA</i> was knocked down with <i>ppl-Gal4</i> in salivary gland and <i>tub-Gal4</i> in brain and wing disc. Different tissues dissected from wandering 3^rd instar larvae of <i>Gal4</i> controls and <i>CdsA RNAi</i> were stained by Nile red (red) or DAPI (blue). Relative tissue sizes were quantified in multiple samples (salivary gland: n = 8; brain and wing disc: n = 10) based on the area occupied. Scale bar (B, C, D): 50 µm.</p

<i>CdsA</i> mutations affect salivary gland fat storage and cell size.

<p>(A) Schematic representation of the <i>CdsA</i> (<i>CG7962</i>) genomic locus. The location within <i>CdsA</i> of two <i>P</i> element insertions, <i>EY08412</i> (for overexpression studies) and <i>GS8005</i> (for mutant clonal analysis) are shown. P1 and P2: primers for RT-PCR in (B). (B) The <i>CdsA^GS8005</i> allele is likely a null. Embryo/1^st instar larval (10–24 hr) RNA of <i>wt</i>, trans-heterozygous or homozygous <i>CdsA^GS8005</i> was analyzed by RT-PCR. Note that there is no detectable <i>CdsA</i> transcript in <i>CdsA^GS8005</i> mutants. <i>Act5C</i> is a control for RT-PCR. (C) A <i>CdsA</i> mutant cell (non-GFP, dashed white circle) is small and contains more neutral lipids compared with neighboring control cells (solid white circle). <i>CdsA</i> mutant salivary gland clones were induced by flp/FRT-mediated recombination during embryogenesis and visualized in wandering 3^rd instar larvae. Blue: DAPI staining for nuclei; red: Nile red staining for neutral lipids; green: Histone::GFP. Histogram: n = 8. Scale bar (C): 50 µm. Error bars (C) represent SEM. (*) P<0.05; (**) P<0.01; (***) P<0.001 (Student's t-test).</p

Loss of function of <i>CdsA</i> does not affect fat cell lipid storage and growth.

<p>(A) Nile red staining of wandering 3^rd instar larval fat body of <i>ppl>GFP</i> control, <i>CdsA RNAi</i>, and <i>CdsA</i> overexpression. Neither silencing nor overexpressing <i>CdsA</i> affects fat body lipid storage. Blue: DAPI staining for nuclei; red: Nile red staining for neutral lipids. (B) Total body fat (normalized to total protein) of wandering 3^rd instar male larvae of <i>ppl>GFP</i> control, <i>CdsA RNAi</i>, and <i>CdsA</i> overexpression. Glyceride assays shown here are representative experiments based on triplicate measurements with a total of ≥15 male larvae per genotype. The level of glyceride is normalized to total proteins. (C) Fat body <i>CdsA</i> mutant cells (non-GFP, dashed white circles) are normal in size and lipid content. Fat body <i>CdsA</i> mutant clones were induced by mitotic recombination 8 hr after egg laying and visualized in wandering 3^rd instar larvae. Blue: DAPI staining for nuclei; red: Nile red staining for neutral lipids; green, Histone::GFP. Histogram: n = 7. (D) tGPH reporter assay in fat body cells. <i>CdsA RNAi</i> leads to reduced membrane-to-cytoplasm ratio of the tGPH GFP fluorescence. Images were all taken with the same exposure time. Note that the total GFP fluorescence is also diminished. Histogram: n = 18. (E) Phospholipid levels of <i>ppl-Gal4</i> control and <i>ppl>CdsA RNAi</i> fat body from wandering 3^rd instar larvae. Note that the PI level in the <i>CdsA RNAi</i> fat body sample is reduced to approximately one third of that in the <i>Gal4</i> control. Assays were done in triplicate. The levels of PA and PI are normalized to total phospholipids. (F) Neither silencing nor overexpressing <i>CdsA</i> affects larval fat body Akt phosphorylation at Ser505. Total Akt and phosphorylated Akt (Ser505) levels were detected by western blotting. α-tubulin (Tub) was used as a loading control. Average and standard deviation of relative band intensity ratio of p-Akt <i>versus</i> total Akt from three replicates is indicated at the top after normalization. Western blot result for one experiment is shown here. Scale bar (A, C, D): 50 µm. Error bars (B, C, D, E) represent SEM. (*) P<0.05; (**) P<0.01; (***) P<0.001 (Student's t-test).</p

A DAG-to-PE route mediated by Bbc may support cell growth in <i>CdsA RNAi</i> fat body.

<p>(A) Fat body <i>Pis</i> mutant cells (marked by absence of RFP, dashed white area) are slightly smaller than neighboring twin-spot wild-type cells. Fat body <i>Pis</i> mutant clones were induced 8 hr after egg laying and visualized in wandering 3^rd instar larvae. Blue: DAPI staining; green: BODIPY staining for neutral lipids; red: Ubi-mRFP, which marks cytosol. Histogram: n = 7. (B) Total PI levels in control and <i>Pis RNAi</i> fat body. The level of PI is normalized to total phospholipids. (C) Nile red staining of fat bodies in larvae with loss of function of insulin pathway components or silencing of <i>CdsA</i> and <i>bbc</i>. Silencing <i>CdsA</i> or <i>bbc</i> alone does not affect fat body cell size, whereas reduced insulin pathway activity dramatically decreases the cell size. Double silencing of <i>CdsA</i> and <i>bbc</i> also significantly reduces fat body cell size. Histogram: n≥94 for each genotype. Blue: DAPI staining for nuclei; red: Nile red staining for neutral lipids. (D) Removing one copy of <i>PI3K</i> or <i>Akt</i> in a <i>ppl>CdsA RNAi</i> background is sufficient to reduce fat body cell size. Histogram: n≥80 for each genotype. Blue: DAPI staining for nuclei; red: Nile red staining for neutral lipids. (E, F) DAG and PE levels obtained by lipid measurements of whole larva, salivary gland or fat body of wandering 3^rd instar larvae of <i>Gal4</i> control, <i>CdsA RNAi</i>, <i>CdsA</i> overexpression and <i>Pis RNAi</i>. <i>CdsA</i> or <i>Pis</i> was knocked down with <i>ppl-Gal4</i> in salivary gland and fat body and <i>tub-Gal4</i> in whole larva. Assays were done in triplicate. Note that the PE level in the <i>CdsA RNAi</i> fat body sample was 47.6% higher than the <i>Gal4</i> control. The levels of DAG and PE are normalized to total phospholipids. (G) Double silencing of <i>CdsA</i> and <i>bbc</i> decreases larval fat body Akt phosphorylation at Ser505. Total Akt and phosphorylated Akt (Ser505) levels were detected by western blotting. α-tubulin (Tub) was used as a loading control. Average and standard deviation of relative band intensity ratio of p-Akt <i>versus</i> total Akt from three replicates is indicated at the top after normalization. The Western blot result from one experiment is shown here. (H) Schematic model depicting the underlying mechanisms of different phenotypes observed in <i>CdsA</i> and <i>Pis</i> mutants. In wild type, <i>de novo</i>-synthesized PI contributes most to the growth of salivary gland cells, while PI from an external source along with DAG, probably from the intestine, contributes most to the growth and fat storage of fat body cells. Both <i>CdsA</i> and <i>Pis</i> mutations lead to ∼80% growth reduction in salivary gland due to the loss of <i>de novo</i>-synthesized PI. In fat body, the loss of <i>de novo</i>-synthesized PI in <i>Pis</i> mutants results in ∼30% reduction of cell growth, while the increase in PE (marked in green) in <i>CdsA</i> mutants compensates for the loss of <i>de novo</i>-synthesized PI. Fat storage in the salivary gland of <i>CdsA</i> mutants increases dramatically. Scale bar (A, C, D): 50 µm. Error bars (A, B, C, D, E, F) represent SEM. (*) P<0.05; (**) P<0.01; (***) P<0.001 (Student's t-test).</p

The insulin pathway genetically interacts with <i>CdsA</i> and affects the transcription level of <i>CdsA</i>.

<p>(A) Perturbation of insulin pathway activity affects salivary gland size and fat storage. Expression of dominant-negative <i>PI3K</i> (<i>PI3K DN</i>) or knockdown of <i>Akt</i> by RNAi leads to lipid accumulation in salivary glands of dramatically reduced size, reminiscent of <i>CdsA RNAi</i>. Blue: DAPI staining for nuclei; red: Nile red staining for neutral lipids. (B, C) Elevation of insulin pathway activity by overexpressing <i>Akt</i> or by expressing constitutively active <i>PI3K (PI3K CA)</i> fully rescues the small salivary gland size phenotype of <i>CdsA RNAi</i>. Note that the massive fat accumulation in <i>CdsA RNAi</i> salivary gland is also partially rescued by <i>PI3K CA</i> expression. Blue: DAPI staining for nuclei; red: Nile red staining for neutral lipids. (D, E) Overexpressing <i>CdsA</i> partially but significantly rescues the small salivary gland phenotype of <i>PI3K DN</i>. Note that the lipid accumulation in <i>PI3K DN</i> can be fully suppressed by <i>CdsA</i> overexpression. Blue: DAPI staining for nuclei; red: Nile red staining for neutral lipids. (F) The insulin pathway positively regulates <i>CdsA</i> expression. Relative <i>CdsA</i> mRNA levels were quantified by qRT-PCR on dissected salivary glands from wandering 3^rd instar larvae of each genotype. Measurements were made in triplicate. (G) The positive feedback loop between the insulin pathway and <i>CdsA</i>. CdsA affects insulin pathway activity by regulating PI/PIP3 levels, while the insulin pathway regulates the <i>CdsA</i> transcription level. Histogram (<i>C</i>, <i>E</i>): n≥8 for each genotype. Scale bar (A, B, D): 50 µm. Error bars (C, E, F) represent SEM. (*) P<0.05; (**) P<0.01; (***) P<0.001 (Student's t-test).</p

Isolation of ATS from <i>abc3</i>Δ appressoria in <i>M. oryzae</i>.

<p>(<b>A</b>) Wild-type <i>S. pombe</i> cells were treated with extracellular fluid (E/F) or appressorial extract (A/E) from the wild-type or <i>abc3</i>Δ <i>M. oryzae</i> strain for 6 h and stained with calcofluor white (CFW). Arrowheads indicate aberrant deposition of septal/cell wall material at the cell tip(s). Bars = 10 µm. (<b>B</b>) Schematic representation of the <i>S. pombe</i> cell-based assay used to guide the purification of ATS and to confirm ATS as an efflux substrate of the Abc3 transporter. Mo<i>ABC3</i> refers to <i>M. oryzae ABC3</i>.</p

Rapid and sensitive profiling of tear wax ester species using high performance liquid chromatography coupled with tandem mass spectrometry

A rapid and sensitive method was developed for quantitative profiling of wax esters (WEs) in human tear lipide. Individual WE species was separated by liquid chromatography and detected by electrospray ionisation mass spectrometry using specific multiple reaction monitoring (MRM) scanning. Palmitoyl palmitate and in-house synthesized wax esters 13C18:1(oleic acid-1,2,3,7,8,9,10-13C7)C26:0 were used as internal standards for quantitation of WEs containing saturated and unsaturated fatty acids (FA), respectively. The limit of detection was approximately 70 nmol/L. The linearity range of the liquid chromatography (LC)-MRM detection for WEs was about three orders of magnitude. Quantitative analyses of 141 individual WE in the human tear lipidome demonstrated that species comprising FA18:1 and FA16:1 each accounted for 47.7% and 24.0% (molar%) of total WE, while fatty alcohols in WEs of human tears ranged from 17 carbons to 32 carbons with predominant species represented by C24, C25 and C26.</p

ATS associates with Tef2 in <i>S. pombe</i> and <i>M. oryzae</i>.

<p>(<b>A</b>) Loss of SpTef2-function simulates ATS effect in <i>S. pombe</i>. Cell wall staining of the wild-type or <i>tef2</i>Δ <i>S. pombe</i> cells using CFW. Red arrowheads depict defective septal/cell wall deposition. Scale bar equals 10 micron. (<b>B</b>) Effect of digoxin on subcellular localization of SpTef2-RFP or Swo1-GFP in <i>S. pombe</i> cells. The strains expressing the indicated fusion proteins were stained with CFW and analysed by epifluorescence microscopy. Arrowheads show distinct aggregates of SpTef2-RFP. Bar = 10 µm. (<b>C</b>) Effect of ATS on localization of RFP-Tef2 in <i>M. oryzae</i> vegetative hyphae (upper panels; Scale Bar = 5 µm) and conidia (middle and lower panels; Bar represents 10 µm) co-stained with DAPI to aid visualization of nuclei. Arrowheads denote aberrant perinuclear aggregates and/or patches of RFP-Tef2. BF, Bright Field.</p