Direct visualization of de novo lipogenesis in single living cells - PubMed (original) (raw)

Direct visualization of de novo lipogenesis in single living cells

Junjie Li et al. Sci Rep. 2014.

Abstract

Increased de novo lipogenesis is being increasingly recognized as a hallmark of cancer. Despite recent advances in fluorescence microscopy, autoradiography and mass spectrometry, direct observation of de novo lipogenesis in living systems remains to be challenging. Here, by coupling stimulated Raman scattering (SRS) microscopy with isotope labeled glucose, we were able to trace the dynamic metabolism of glucose in single living cells with high spatial-temporal resolution. As the first direct visualization, we observed that glucose was largely utilized for lipid synthesis in pancreatic cancer cells, which occurs at a much lower rate in immortalized normal pancreatic epithelial cells. By inhibition of glycolysis and fatty acid synthase (FAS), the key enzyme for fatty acid synthesis, we confirmed the deuterium labeled lipids in cancer cells were from de novo lipid synthesis. Interestingly, we also found that prostate cancer cells exhibit relatively lower level of de novo lipogenesis, but higher fatty acid uptake compared to pancreatic cancer cells. Together, our results demonstrate a valuable tool to study dynamic lipid metabolism in cancer and other disorders.

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Figures

Figure 1

Figure 1. Isotope labeled glucose-d7 is a tracer for de-novo lipogenesis.

(a). Scheme of glucose-derived de-novo lipid synthesis in mammalian cells. (b). Raman spectrum of 1.0 M glucose-d7 aqueous solution. A broad and unique peak around 2120 cm−1 from C–D bond vibration was observed. Inset: chemical structure of glucose-d7. (c). Linear dependence of SRS signal on glucose-d7 concentration. The detection limit of our SRS imaging is around 50 mM. Inset: SRS image of 1.0 M glucose-d7 solution at the interface of solution and air.

Figure 2

Figure 2. Glucose-d7 is utilized for lipid synthesis in pancreatic cancer PANC1 cells.

PANC1 cells were incubated with (a). 25 mM glucose-d7 or (b). 25 mM glucose in glucose-free DMEM media supplemented with 10% FBS for 3 days. SRS imaging was taken at C–D vibration (~2120 cm−1), off-resonance (~2600 cm−1) or C–H vibration (~2850 cm−1). (c). Raman spectra acquired from lipid droplets in control cells or glucose-d7 treated cells. (d). Nontoxicity test of glucose-d7 compared to regular glucose. Cell viability was determined by MTT assay. Data was represented as Mean + SD with n = 6 for each group.

Figure 3

Figure 3. Dynamics of glucose-d7 derived lipogenesis in PANC1 cells.

(a). Monitoring de-novo lipogenesis in PANC1 cells over time by SRS imaging at C–D and C–H vibration. Inset of image at 72 h: zoom in image of marked area showing the donut-shaped newly synthesized LDs. (b). Quantification of SRS signal at C–D vibration per cell. Data were shown as Mean ± SD. N = 5 for each group. (c). SRS imaging of PANC1 cells incubated with 10 mM 2-DG and 25 mM glucose-d7 for 3 days. (d). SRS imaging of PANC1 cells incubated with glucose-d7 for 3 days and treated with DMSO or 20 μM FAS inhibitor C75 for 24 hours. (e). SRS imaging of PANC1 cells stably transfected with FAS shRNA and incubated with glucose-d7 for 3 days.

Figure 4

Figure 4. Increased lipogenesis in pancreatic cancer cells than normal pancreatic cells.

(a). Normal immortalized pancreatic epithelial HPDE6 cells, and (c). pancreatic cancer PANC1 cells were treated with 25 mM glucose-d7 in glucose-free DMEM media supplemented without or with 10% FBS for 3 days. SRS imaging at C–D and C–H vibration were taken. The ratio of C–D/C–H was used to analyze the level of de-novo lipogenesis. Quantitative analysis of de-novo lipogenesis level in (b). HPDE6 and (d). PANC1 cells. The ratio of C–D signal to C–H signal intensity on single lipid droplets was measured. At least 10 lipid droplets were analyzed for each sample. The data was represented by Mean + SD. *** indicates p < 0.001 by student T test.

Figure 5

Figure 5. Lipogenesis and fatty acid uptake in prostate and pancreatic cells.

(a). Glucose-d7 derived lipogenesis were measured in prostate cell lines, including normal epithelial RWPE1 cell and cancerous LNCaP, PC3 cells by SRS imaging at C–D and C–H vibration. (b). Quantitative analysis of de-novo lipogenesis level in prostate cell lines. Data were shown as mean + SD. N ≥ 10 for each group. *** indicates p < 0.001 by student T test. (c). SRS imaging of prostate cells and (d). pancreatic cells treated with 100 μM palmitic acid-d31 for 24 hours.

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