Study provides the first visual evidence of lipid breakdown at the single-droplet level in living cells

A research team has developed a fluorescent probe that allows scientists to visualise how individual lipid droplets break down inside living cells in real-time. The probe changes its fluorescence properties depending on the chemical composition of each droplet, which allows researchers to observe not only their location within cells, but also their metabolic activity during lipid breakdown. The findings may contribute to the development of new strategies to treat metabolic diseases such as obesity and diabetes, as well as cancers associated with abnormal lipid metabolism.

LipiPB Red shows longer fluorescence lifetimes in stable lipid droplets (red) and shorter lifetimes as they undergo degradation (blue). This probe revealed that lipid droplets sequentially degrade, where lipolysis precedes lipophagy (Credit: Issey Takahashi, Nagoya University).

“Lipid droplets are cellular organelles that not only store excess lipids but also play critical roles in lipid metabolism. However, understanding how individual droplets function has been challenging,” explained Professor Shigehiro Yamaguchi, from the Institute of Transformative Bio-Molecules (ITbM) at Nagoya University. “Traditional methods require destroying cells and can only measure the average behavior across many droplets. This approach misses important differences and leaves key questions about how cells regulate their energy reserves unanswered. Our organic fluorescent probe LipiPB Red fills this gap, allowing us to observe functional differences among individual droplets for the first time.”

The fluorescence lifetime of LipiPB Red - how long it continues to emit light after absorbing light energy - depends on the type of lipid molecules inside each droplet. When triglycerides (the main storage form of neutral lipids) are the major component, the probe shows longer fluorescence lifetimes. As they are enzymatically degraded into diglycerides (partially hydrolyzed forms of triglycerides), the fluorescence lifetime becomes shorter. By monitoring these changes, the researchers could identify which lipid droplets were actively breaking down.

Using LipiPB Red, the team observed that lipid droplets in liver cancer cells break down at markedly different rates, even within the same cell, a phenomenon not seen in other cell types. Scientists had assumed that all droplets within a cell would behave similarly, which was true for other cell types. Further experiments revealed that an enzyme called ATGL (adipose triglyceride lipase) is responsible for these differences. 

“ATGL is already known to be important for converting triglycerides to diglycerides during lipid droplet breakdown, but we found that its activity differs dramatically among individual droplets within the same cell,” said Professor Masayasu Taki from the Institute for Glyco-core Research (iGCORE), Gifu University.  “When ATGL was inhibited, the differences disappeared, confirming its central role. These findings indicate that liver cancer cells metabolise lipids in a uniquely heterogeneous manner, which may be linked to their abnormal energy regulation.”

The team also achieved the first direct visualization of the sequential lipid breakdown process in real time. They observed that lipid droplets are first degraded enzymatically through lipolysis, followed by further breakdown via autophagic pathways (lipophagy). Although how lipid droplet breakdown is regulated remains controversial, LipiPB Red allows direct observation of the process within living cells.

LipiPB Red also provides a powerful new tool to study fundamental questions about cellular energy regulation and could reveal how cells make decisions about when and how to access their energy reserves during stress, exercise, or disease.

“We can now study how different treatments or conditions affect cellular lipid metabolism at a level of detail that was not possible before. This technology will advance our understanding of lipid metabolism and could help develop new therapeutic approaches for metabolic disorders and cancer,” added Taki.

The findings were published in the paper, ‘Single-cell fluorescence analysis of lipid droplet compositional dynamics during triacylglycerol catabolism’, published in the Journal of the American Chemical Society. To access this paper, please click here