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

LipiDye-M is a C12 fatty acid mimic labeled with a novel solvatochromic dye. As LipiDye-M exhibits green-to-red fluorescence depending on its lipid structure and its localization, LipiDye-M can trace status of cellular fatty acid uptake and lipid metabolism in cells. LipiDye-M is a powerful tool for both basic research and pharmaceutical research for lipid metabolism.

Overview of intracellular metabolic pathway of LipiDye-M (Left) and three color imaging (Right)
LipiDye-M is a novel solvatochromic dye-labeled fatty acid and can be taken up from extracellular space by FA-transporters. LipiDye-M exhibits green-to-red fluorescence depending on its lipid structure and its localization. LipiDye-M allows to perform three-color imaging (green, yellow and red) by merging images from a green channel and red channel.

• MEMO
• Feature
• Fluorescent property and recommended condition
• Reference
• Application data
• Product information

MEMO

 

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Feature

-       LipiDye-M exhibits green-to-red fluorescence depending on its lipid structure and its localization

-       LipiDye-M can visualize status of fatty acid metabolism by confocal fluorescent microscopy

-       Likely to native fatty acids, LipiDye-M can also be taken up to cells by FA-transporters and converted into many types of lipids, including acyl-CoA, phospholipids, DAGs, TAGs, and degraded to small metabolites by the mitochondrial FAO pathway.

-       LipiDye-M emits green fluorescence in high polarity (cytosol), yellow fluorescence in moderate polarity (organelle membranes) and red fluorescence in low polarity (lipid droplets).

-       LipiDye-M allows to perform three-color imaging (green, yellow and red) by merging images from a green channel (Ex. 450-490 nm / Em. 490-540 nm) and red channel (Ex. 550-600 nm / Em. 570-620 nm)

 

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Fluorescent property and recommended condition

Absorption spectrum (left) and emission spectrum (right) in PBS, phospholipid bilayers of large unilamellar vesicles (LUVs), and soybean oil. PBS, phospholipid LUV, and soybean oil are mimicked for cytosol, organelle membranes, and lipid droplets, respectively. Based on the spectral features, the following two channels: a green channel (Ex. 450-490 nm, recommended laser 473 nm / Em.490-540 nm) and a red channel (Ex. 550-600 nm, recommended laser 559 nm / Em. 570-620 nm) are recommended to distinguish lipid metabolites. To properly distinguish lipid metabolites, confocal laser microscopy is highly recommended.

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Reference

1. Kajiwara et al.,Nat. Commun., 13, 2533 (2022) A negative-solvatochromic fluorescent probe for visualizing intracellular distributions of fatty acid metabolites.

 

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Application data

Distribution of LipiDye-M metabolites in live adipocytes

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.

Distribution of LipiDye-M metabolites in live HepG2

(Left) HepG2 cells were treated with oleic acid/palmitic acid in a complete medium containing 10% FBS to mature lipid droplets. Afterward, the culture media was exchanged with HBSS containing LipiDye-M (5 µM), the cells were incubated for 1 hour and observed by 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). Under serum-starved conditions, the merged image showed LipiDye-M effectively incorporated into the cells, and its metabolites were distributed in not only the cytosol (green), ER (yellow), and lipid droplets (red) but also mitochondria as yellow.

(Right) To investigate metabolites of LipiDye-M in the cells, lipid fractions were biochemically extracted from the cells and separated by thin-layer chromatography (TLC) with fluorescent detection. Many metabolites of LipiDye-M were detected in the lipid extracts from HepG2 and were rationalized as esterified products (TAG or cholesteryl ester), phospholipids and FAO metabolites.

Effect of FA metabolism inhibitors on the distribution of LipiDye-M metabolites

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.  

 

Effect of inhibitors for lipid degradation pathway on the distribution of LipiDye-M metabolites

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. Under the treatment of lipolysis inhibitor, overall red signals in the merged image significantly increased, and the Green/Red ratio was clearly decreased in the ratio image and the pixel plot. This data indicate lipids were abnormally accumulated in lipid droplets and organelle membranes by suppression of the lipolysis pathway. Detailed information about the ratiometr ic analysis is described in the Reference paper.

Reference data: Cytotoxicity of LipiDye-M

To observe the cytotoxicity of LipiDye-M, HepG2 cells were treated with various concentrations of LipiDye-M for 24 hours. Afterward, cell viability was assessed by MTT assay. Under 10 µM LipiDye-M shows little effect on HepG2 viability. However, 20 µM LipiDye-M exhibits weak cytotoxicity (~20%) on HepG2 cells.

 

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Product information