Fatty Acid Oxidation (FAO) activity detection / assay reagent FAOBlue^™(Fatty Acid Oxidation Detection Reagent)

Image of FAOBlue^™ reagent

FAOBlue^™ is a fatty acid analog consisting of a nonanoic acid (C9) with a blue fluorescence dye coumarin-derivative at the end of the carbon chain, and further its carboxylic acid is protected with acetoxymethyl ester to improve cell membrane permeability. Although FAOBlue^™ contains a coumarin derivative, it shows no-fluorescence excited by 405 nm before metabolization by FAO.

Since this reagent uses 405 nm wavelength as excitation light, autofluorescence may be observed depending on samples. Negative control (without this reagent) for each experiment is recommended to confirm cell-derived autofluorescence signals. In particular, if a dot-like fluorescent signals are observed during microscopic observation, they may be caused by autofluorescence.

FAOBlue^™ is converted to a coumarin derivative by FAO, resulting in a long wavelength shift of the absorption maximum from 350 nm to 405 nm. Therefore, under 405 nm excitation, FAOBlue^™ doesn’t show fluorescence, and blue fluorescence is observed only when the coumarin derivative is released by FAO.

Four types of cancer cells were incubated with FAOBlue^™ and fluorescence was observed after a certain time of incubation. All cell lines showed blue fluorescence, but pre-treatment of etomoxir clearly decreased fluorescent intensities. These results indicated the blue fluorescence was derived from FAO activity in the cells.

HepG2 cells were pre-incubated with a lipid metabolism enhancer AICAR^*(200 μM, 3 hours) or a partial FAO inhibitor Ranolazine (200 μM, 12 hours), and then incubated with FAOBlue^™(5 μM) for 30 min. Compared with control cells, pretreatment with AICAR significantly increased blue fluorescence intensity. On the other hand, pretreatment with the partial FAO inhibitor ranolazine clearly decreased blue fluorescence intensity.
＊AICAR: AMPK (AMP-activated protein kinase) activator, which activates lipid metabolism.

FAOBlue^™ can be used to quantitatively analyze the effects of drugs on FAOs. After HepG2 cells were pre-incubated with ND-630 (acetyl-CoA carboxylase inhibitor), a drug candidate for the treatment of metabolic dysfunction associated steatotic liver disease (MASLD), for 4 hours, the cells were incubated with FAOBlue^™(5 μM) for 30 min. Evaluation of blue fluorescence intensity by confocal laser microscopy showed a ND630 concentration-dependent increase in fluorescence intensity, indicating that FAO activity is enhanced by ND-630.

Metabolic dysfunction associated steatohepatitis (MASH) is a typical disease which shows low metabolic activity of FAs. After control healthy mice and MASH model mice were orally administered with bezafibrate, a therapeutic agent of MASH, primary hepatocytes were isolated from livers of control mice and MASH model mice. Primary hepatocytes were treated with 5 uM FAOBlue^™ for 30 min and fluorescence imaging was performed. Compared with control cells, MASH model mouse-derived hepatocytes showed low FAO activity. On the other hand, Bezafibrate dramatically recovered FAO activity of hepatocytes isolated from MASH model mouse. This reagent is a powerful tool to estimate drug effects and efficiency on FAO activity.

786-O cells were seeded in 96 well and culture for 1 day. Confluent cells were treated with various drugs in serum-free DMEM medium for 20 hours. After wash cells with HBS, cells were treated with final 10 uM of FAOBlue^™ in HBS for 2 hours. Without washing cells, fluorescent intensity (Ex 420±5 / Em 460±10) by the fluorescent plate leader with a transparent mode. Fluorescent signals were normalized by the background signal of 10 uM FAOBlue^™ without cells. A mitochondrial FAO inhibitor, etomoxir, clearly suppress blue fluorescence signal, indicating that FAOBlue^™ detected fluorescence signals dependent on mitochondrial FAO activity. [Drug treatment: 10 uM Etomoxir (mitochondrial FAO inhibitor), 500 uM AICAR (AMPK activator), 5 uM 2-bromopalmitate (2-BP; broad lipid metabolism inhibitor)]