Visualizing lipid metabolism on live cells by three color fluorescent imaging LipiDye™-M: Lipid Metabolism Tracer

LipiDye™-M is a solvatochromic dye-labeled fatty acid. After it is internalized through fatty acid transporters, it undergoes lipid metabolism in the cell, and its fluorescence changes as its localization changes. Green-fluorescence and red-fluorescence pictures are obtained by imaging, and by superimposing them, the state of lipid metabolism can be distinguished by three colors.

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In order to properly observe the difference in environmental response of LipiDye™-M, it is necessary to carefully consider the excitation wavelength and fluorescence wavelength. Confocal microscope is recommended. Please consider the excitation light and detection filter with reference to the spectrum below.

excitation/fluorescence spectrum of LipiDye™-M in PBS, phospholipid Liposomes, and soybean oil

 Uptake in adipocytes    Uptake in HepG2 cells    Changes in lipid metabolism by drug treatment    Effect of lipid degradation pathway inhibitors    Cytotoxicity 

Adipocytes derived from 3T3-L1 cells were treated with LipiDye™-M (5 µM) and observed after 1 min or 30 min with confocal fluorescent microscopy without a wash step. Confocal images were recorded in the green channel (Ex. 473 nm/Em. 490-540 nm) and red channel (Ex. 559 nm/Em 570-620 nm). The images captured at 1 min shows strong green fluorescence intensity from cytosol and organelle membrane and weak red intensity from lipid droplets. This indicates major metabolites of LipiDye™-M are free-FA, and acyl-CoA and LipiDye™-M are insufficient to incorporate into TAGs. After 30 min, the intensity of the green channel decreased, and the red fluorescent signal from lipid droplets clearly increased. These images suggest LipiDye™-M was sufficiently converted into TAG lipids and accumulated into lipid droplets. Under treatment with a DGAT inhibitor to block TAG synthesis, a slight red signal is detected from lipid droplets, and the yellow signal of organelle membranes on the merged image dramatically increased. This result indicates DGAT inhibitor blocks TAG biosynthesis on ER membrane, and metabolites of LipiDye™-M were highly accumulated in organelle membranes as DAG species.

HepG2 cells were treated with FA metabolism inhibitors, acyl-CoA synthase (ACS) inhibitor (Triacsin C, 5 µM) or DGAT1 inhibitor (T863, 20 µM), in complete medium for 18 hours. Afterward, the media was replaced with HBSS containing 5 µM LipiDye™-M, 0.5 mM oleic acid, and the indicated inhibitor. The cells were incubated for 6 hours and observed. Confocal images were recorded in the green channel (Ex. 473 nm/Em 490-540 nm) and the red channel (Ex. 559 nm/Em 570-620 nm). Merged images are shown below on the left, and proposed metabolic pathways under treatment of each inhibitor are shown on the right. In the control cell, strong red signals from lipid droplets were observed. Under treatment with ACS inhibitor, both red and yellow signals dramatically decreased, and green signals increased. This result indicates ACS inhibitor suppresses the conversion of free-FA to acyl-CoA, and the free-FA form of LipiDye™-M is accumulated in the cytosol and organelles. Under treatment of DGAT inhibitor, yellow signals clearly increased, while a little red signal from lipid droplet was observed. This data shows DGAT1 inhibitor blocked the conversion of DAG to TAG, and excess DAG was accumulated in the ER.

HepG2 cells were cultured in HBSS containing LipiDye™-M (5 µM) and autophagy inhibitor (50 nM Bafilomycin A1) or lipolysis (100 µM DEUP) for 6 hours. Afterward, confocal images were recorded in the green channel (Ex. 473 nm/Em 490-540 nm) and red channel (Ex. 559 nm/Em 570-620 nm). Both images merged (Green + Red; left), and ratio images (Green/Red; right) were calculated. In the control condition, mitochondria (green) and lipid droplets (red) were mainly observed in the merged image. Under the treatment of autophagy inhibitor, vesicle-structure indicating autophagosomes were detected by yellow color in the merged image. This data indicates lipid degradation by lipophagy was suppressed by the autophagy inhibitor.

* Detailed information about the ratiometric analysis is described in ☞ the original paper.

Kajiwara, K., et al., "A negative-solvatochromic fluorescent probe for visualizing intracellular distributions of fatty acid metabolites.", Nat. Commun., 13(1), 2533 (2022). [PMID:35534485]