Acylglycerol Recycling from Triacylglycerol to Phospholipid, Not Lipase Activity, Is Defective in Neutral Lipid Storage Disease Fibroblasts

Neutral lipid storage disease (NLSD) is an autosomal recessive disorder in which excess triacylglycerol (TG) accumulates in most cells. Although it has been hypothesized that the TG accumulation is caused by a functional defect in cytosolic lipase activity, we were able to expose TG hydrolysis in NLSD cells by using triacsin C, an inhibitor of acyl-CoA synthetase that blocks the reincorporation of hydrolyzed fatty acids into glycerolipids. Our data suggest that TG lipolysis in NLSD cells is masked by rapid TG resynthesis, occurring because released acylglycerols cannot be used for phospholipid synthesis. In uptake studies, triacsin C blocked the incorporation of [3H]glycerol into glycerolipids, incorporation of [14C]oleate into TG, but not incorporation of [14C]oleate into phospholipid. Thus, the drug inhibited both de novo synthesis of glycerolipids via the glycerol-3-phosphate pathway and the synthesis of TG from diacylglycerol. The drug did not appear to block reacylation of lysophospholipids. Triacsin C caused a loss of about 60% of the TG mass from both NLSD and oleate-loaded control cells. Rates of TG lipolysis were similar in NLSD cells and oleate-loaded control cells labeled with [6-(7-nitro-2,1,3-benzoxadiazol-4-yl)-amino]hexanoic acid or labeled with [14C]oleate or [3H]glycerol and chased in the presence of triacsin C. During a 96-h chase, [14C]oleate reincorporation into the different phospholipid species increased only in control cells. Similar results were observed when NLSD, and control cells were chased after labeling with [3H]glycerol. These data strongly suggest that normal human fibroblasts mobilize stored TG for phospholipid synthesis and that recycling to PC occurs via a TG-derived mono- or diacylglycerol intermediate. Normal recycling to phosphatidylethanolamine may primarily involve TG-derived acyl groups rather than an acylglycerol precursor. NLSD cells appear to have a block in this recycling pathway with the result that both hydrolyzed fatty acids and the acylglycerol backbone are re-esterified to form TG. Because the NLSD phenotype includes ichthyosis, fatty liver, myopathy, cardiomyopathy, and mental retardation, the recycling pathway appears to be critical for the normal function of skin, liver, muscle, heart, and the central nervous system

StearoylCoA Desaturase-5: A Novel Regulator of Neuronal Cell Proliferation and Differentiation

Recent studies have demonstrated that human stearoylCoA desaturase-1 (SCD1), a Δ9-desaturase that converts saturated fatty acids (SFA) into monounsaturated fatty acids, controls the rate of lipogenesis, cell proliferation and tumorigenic capacity in cancer cells. However, the biological function of stearoylCoA desaturase-5 (SCD5), a second isoform of human SCD that is highly expressed in brain, as well as its potential role in human disease, remains unknown. In this study we report that the constitutive overexpression of human SCD5 in mouse Neuro2a cells, a widely used cell model of neuronal growth and differentiation, displayed a greater n-7 MUFA-to-SFA ratio in cell lipids compared to empty-vector transfected cells (controls). De novo synthesis of phosphatidylcholine and cholesterolesters was increased whereas phosphatidylethanolamine and triacylglycerol formation was reduced in SCD5-expressing cells with respect to their controls, suggesting a differential use of SCD5 products for lipogenic reactions. We also observed that SCD5 expression markedly accelerated the rate of cell proliferation and suppressed the induction of neurite outgrowth, a typical marker of neuronal differentiation, by retinoic acid indicating that the desaturase plays a key role in the mechanisms of cell division and differentiation. Critical signal transduction pathways that are known to modulate these processes, such epidermal growth factor receptor (EGFR)Akt/ERK and Wnt, were affected by SCD5 expression. Epidermal growth factor-induced phosphorylation of EGFR, Akt and ERK was markedly blunted in SCD5-expressing cells. Furthermore, the activity of canonical Wnt was reduced whereas the non-canonical Wnt was increased by the presence of SCD5 activity. Finally, SCD5 expression increased the secretion of recombinant Wnt5a, a non-canonical Wnt, whereas it reduced the cellular and secreted levels of canonical Wnt7b. Our data suggest that, by a coordinated modulation of key lipogenic pathways and transduction signaling cascades, SCD5 participates in the regulation of neuronal cell growth and differentiation

Inhibition of StearoylCoA Desaturase Activity Blocks Cell Cycle Progression and Induces Programmed Cell Death in Lung Cancer Cells

Lung cancer is the most frequent form of cancer. The survival rate for patients with metastatic lung cancer is ∼5%, hence alternative therapeutic strategies to treat this disease are critically needed. Recent studies suggest that lipid biosynthetic pathways, particularly fatty acid synthesis and desaturation, are promising molecular targets for cancer therapy. We have previously reported that inhibition of stearoylCoA desaturase-1 (SCD1), the enzyme that produces monounsaturated fatty acids (MUFA), impairs lung cancer cell proliferation, survival and invasiveness, and dramatically reduces tumor formation in mice. In this report, we show that inhibition of SCD activity in human lung cancer cells with the small molecule SCD inhibitor CVT-11127 reduced lipid synthesis and impaired proliferation by blocking the progression of cell cycle through the G1/S boundary and by triggering programmed cell death. These alterations resulting from SCD blockade were fully reversed by either oleic (18:1n-9), palmitoleic acid (16:1n-7) or cis-vaccenic acid (18:1n-7) demonstrating that cis-MUFA are key molecules for cancer cell proliferation. Additionally, co-treatment of cells with CVT-11127 and CP-640186, a specific acetylCoA carboxylase (ACC) inhibitor, did not potentiate the growth inhibitory effect of these compounds, suggesting that inhibition of ACC or SCD1 affects a similar target critical for cell proliferation, likely MUFA, the common fatty acid product in the pathway. This hypothesis was further reinforced by the observation that exogenous oleic acid reverses the anti-growth effect of SCD and ACC inhibitors. Finally, exogenous oleic acid restored the globally decreased levels of cell lipids in cells undergoing a blockade of SCD activity, indicating that active lipid synthesis is required for the fatty acid-mediated restoration of proliferation in SCD1-inhibited cells. Altogether, these observations suggest that SCD1 controls cell cycle progression and apoptosis and, consequently, the overall rate of proliferation in cancer cells through MUFA-mediated activation of lipid synthesis

Inhibition of StearoylCoA Desaturase-1 Inactivates Acetyl-CoA Carboxylase and Impairs Proliferation in Cancer Cells: Role of AMPK

Cancer cells activate the biosynthesis of saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) in order to sustain an increasing demand for phospholipids with appropriate acyl composition during cell replication. We have previously shown that a stable knockdown of stearoyl-CoA desaturase 1 (SCD1), the main Δ9-desaturase that converts SFA into MUFA, in cancer cells decreases the rate of lipogenesis, reduces proliferation and in vitro invasiveness, and dramatically impairs tumor formation and growth. Here we report that pharmacological inhibition of SCD1 with a novel small molecule in cancer cells promoted the activation of AMP-activated kinase (AMPK) and the subsequent reduction of acetylCoA carboxylase activity, with a concomitant inhibition of glucose-mediated lipogenesis. The pharmacological inhibition of AMPK further decreased proliferation of SCD1-depleted cells, whereas AMPK activation restored proliferation to control levels. Addition of supraphysiological concentrations of glucose or pyruvate, the end product of glycolysis, did not reverse the low proliferation rate of SCD1-ablated cancer cells. Our data suggest that cancer cells require active SCD1 to control the rate of glucose-mediated lipogenesis, and that when SCD1 activity is impaired cells downregulate SFA synthesis via AMPK-mediated inactivation of acetyl-CoA carboxylase, thus preventing the harmful effects of SFA accumulation

Inhibition of SCD1 upregulates AMPK phosphorylation and activity.

<p>A549 and H1299 cells were incubated with 10 µM and 5 µM CVT-11127 (CVT) respectively, or DMSO for 24 h. The levels of phospho-ACC (Ser79) and phospho-AMPKα (Thr172) were determined by Western Blot in CVT-11127-treated cells (A–B) and SCD1-deficient A549 cells (C) and normalized to β-actin content. Cells treated with 20 µM Compound C (CpC) were included as a control. D, regulation of ACC activity by fatty acids. SCD1-deficient (hSCDas) and control A549 cells were serum-starved (0.1% FBS) for 24 h and incubated for 30 min in serum-depleted media with or without 100 µM palmitic (Pal) or oleic (Ole) acid complexed with 0.5% w/v fatty acid-free bovine serum albumin (BSA). Cellular levels of phospho-ACC (Ser79), phospho-AMPK and β-actin were determined by Western blot. Values represent the mean±S.D. of triplicate determinations. *, p<0.05 or less, by Student's t test.</p

Inhibition of SCD activity with CVT-11127 impairs the proliferation of H460 cancer cells but not normal human fibroblasts.

<p>Cell growth was determined in AG01518 normal human fibroblasts (<b>A</b>) in presence of 1 and 2 µM CVT-11127 in 10%FBS DMEM or vehicle for 96 h. H460 cells (<b>B</b>) were treated with 1 µM CVT-11127 or vehicle for 96 h. Cell proliferation rate was assessed by Crystal violet staining. <b>*</b>, p<0.05 or less, vs DMSO; by Student's t test.</p

A novel small molecule inhibitor of SCD activity reduces cell proliferation in human lung and breast cancer cells.

<p>A549 (A) and H1299 (B) cells were incubated with with different concentrations of CVT-11127 (CVT) or DMSO vehicle for 24 h, as described, and cell proliferation was determined by Crystal violet assay. For a similar analysis, H460 cells (C) were treated with 1 µm CVT-11127 for 24 h. For the determination of DNA synthesis, A549 (D) and H460 (E) cells were incubated with 10 µM and 5 µM CVT-11127 or vehicle for 24 h and pulsed with [³H]thymidine (1 µCi/dish) for 2 h at 37°C. Total [³H]-labeled DNA was precipitated, radioactivity was quantified in a scintillation counter and normalized to protein concentration. F, MCF-7 and MDA-MB-231 breast cancer cells, and WS-1 human skin fibroblasts were incubated with 10 µM CVT-11127 (CVT) or DMSO vehicle for 24 h and cell proliferation was assessed by crystal violet staining method. E, H460 cells were incubated for 48 h with 1 µM CVT in presence of 1, 10, 50 and 100 µM sodium oleate complexed with BSA (1∶2 BSA:fatty acid ratio). Cell incubated with DMSO vehicle were considered the control group. Cell growth was estimated by crystal violet staining method. Values represent the mean±S.D. of triplicate determinations. *, p<0.05 or less vs control, by Student's t test.</p

de novo lipid synthesis from glucose is impaired upon SCD inhibition.

<p>A549 cells (A) and H1299 cells (B) were incubated with 10 µM and 1 µM CVT-11127 (CVT), respectively, or DMSO for 24 h. Cells were then pulsed with 1 µCi D-[U-¹⁴C]glucose for up to 24 h. C, A549 cells with a stable knockdown in SCD1 expression (hSCDas) and mock-transfected control cells were subjected to a similar incubation with [¹⁴C]glucose. Cellular lipids were extracted and incorporation of [¹⁴C]glucose into total lipids was quantified by scintillation counting and normalized to protein concentration. D, basal glucose uptake was assayed in H1299 cells treated with 1 µM CVT or vehicle for 24 h by estimating the uptake of [³H]deoxyglucose. E, H1299 cells were incubated with [¹⁴C]glucose in presence or absence of 1 µM CVT for 6 h and levels of total [¹⁴C]fatty acids, as well as radiolabeled SFA and MUFA (upper panel), were determined by argentation TLC as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006812#s2" target="_blank">Materials and methods</a>. F, the rate of lipid synthesis in H1299 cells was assessed by incubation with 1 µM CVT or vehicle and 0.5 µCi/dish of [¹⁴C]acetate for 24 h. Lipids were extracted and radioactivity of total lipids was determined by scintillation counting. Values represent the mean±S.D. of triplicate determinations. *, p<0.05 or less, by Student's t test.</p