Fluorescence detection of metabolic activity of the fatty acid beta oxidation pathway in living cells

Uchinomiya, Shohei; Matsumoto, Naoya; Kamoda, Koichiro; Kawagoe, Ryosuke; Tsuruta, Akito; Ohdo, Shigehiro; Ojida, Akio

doi:10.1039/c9cc09993j

Cited by 34 publications

(26 citation statements)

References 22 publications

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“…3a,b). FAO activity in cultured HepG2 cells, analyzed using a quenched fluorescent probe that is activated by the sequential enzyme reactions of FAO 36 , also revealed no changes in FAO in lipophagy-activated cells (Supplementary Fig. 5c).…”

Section: Resultsmentioning

confidence: 95%

Liver lipophagy ameliorates nonalcoholic steatohepatitis through lysosomal lipid exocytosis

Minami

Hoshino

Higuchi

et al. 2022

Preprint

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Nonalcoholic steatohepatitis (NASH) is a progressive disorder with aberrant lipid accumulation and subsequent inflammatory and profibrotic response. Therapeutic efforts at lipid reduction via increasing cytoplasmic lipolysis unfortunately worsens hepatitis due to toxicity of liberated fatty acid. An alternative approach could be lipid reduction through autophagic disposal, i.e., lipophagy. We engineered a synthetic adaptor protein to induce lipophagy, combining a lipid droplet-targeting signal with optimized LC3-interacting domain. Activating hepatocyte lipophagy in vivo strongly mitigated both steatosis and hepatitis in a diet-induced mouse NASH model. Mechanistically, activated lipophagy promoted the excretion of lipid from hepatocytes via lysosomal exocytosis, thereby suppressing harmful intracellular accumulation of nonesterified fatty acid. A high-content compound screen identified alpelisib and digoxin, clinically-approved compounds, as effective activators of lipophagy. Administration of alpelisib or digoxin in vivo strongly inhibited the transition to steatohepatitis. These data thus identify lipophagy as a promising therapeutic approach to prevent NASH progression.

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Section: Resultsmentioning

confidence: 95%

Liver lipophagy ameliorates nonalcoholic steatohepatitis through lysosomal lipid exocytosis

Minami

Hoshino

Higuchi

et al. 2022

Preprint

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“…β-oxidation of FAs in the mitochondrial matrix is an essential metabolic process for producing energy. However, fluorophores often interfere with the β-oxidation process by causing the fluorescent FAs not to be recognized as substrates or greatly slowing the reaction rate compared to that of natural FAs 11 , 13 . As intense signals of the AP-C12 metabolites were observed in the mitochondria, we can infer that AP-C12 is effectively consumed by β-oxidation in the mitochondrial matrix.…”

Section: Resultsmentioning

confidence: 99%

“…In contrast to the environment-insensitive BODIPY probe, FA analogues with a nitrobenzoxadiazole fluorophore are environment-sensitive, emitting weak fluorescence in aqueous media and intense fluorescence in non-polar environments 12 . Recently, a new type of fluorescent FA that emits strong fluorescence upon completion of β-oxidation has been developed 13 . Although these fluorescent FAs are promising molecular probes that provide valuable biological insights, probes for more advanced applications in the metabolic analysis of FAs demand the following characteristics: (1) A lack of interference with the metabolic pathways of FAs, including β-oxidation in mitochondria, (2) high sensitivity to the local environment polarity of the organelles in which FA metabolites are present, and (3) a neutral charge, to avoid organelle-specific localization 15 .…”

Section: Introductionmentioning

confidence: 99%

A negative-solvatochromic fluorescent probe for visualizing intracellular distributions of fatty acid metabolites

et al. 2022

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Metabolic distribution of fatty acid to organelles is an essential biological process for energy homeostasis as well as for the maintenance of membrane integrity, and the metabolic pathways are strictly regulated in response to environmental stimuli. Herein, we report a fluorescent fatty acid probe, which bears an azapyrene dye that changes its absorption and emission features depending on the microenvironment polarity of the organelle into which it is transported. Owing to the environmental sensitivity of this dye, the distribution of the metabolically incorporated probe in non-polar lipid droplets, medium-polarity membranes, and the polar aqueous regions, can be visualized in different colors. Based on density scatter plots of the fluorophore, we demonstrate that the degradation of triacylglycerols in lipid droplets occurs predominantly via lipolysis rather than lipophagy in nutrition-starved hepatocytes. This tool can thus be expected to significantly advance our understanding of the lipid metabolism in living organisms.

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“…There was no difference in the amount of any kind of triglycerides, which are sources of acyl-carnitine (Fig EV2B). Then, we quantified the fatty acid oxidation activity in HeLa cells upon osmotic stress using FAOBlue, the fluorescence of which reflects the β-oxidation activity in live cells (Uchinomiya et al , 2020). FAOBlue fluorescence intensity, which was abolished by etomoxir (an inhibitor of carnitine palmitoyl transferase 1 (CPT1)), was attenuated under hyperosmotic stress (Fig 2E).…”

Section: Resultsmentioning

confidence: 99%

Mitochondria directly sense osmotic stress to trigger rapid metabolic remodeling via PDH regulation

Ikizawa

Ikeda

Arita

et al. 2022

Preprint

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A high-salt diet significantly impacts various diseases, including cancer and immune diseases. Recent studies suggest that the high-salt/hyperosmotic environment in the body may alter the chronic properties of cancer and immune cells in the disease context. However, little is known about the acute metabolic changes in hyperosmotic stress. Here, we found that hyperosmotic stress for a few minutes induces Warburg-like metabolic remodeling in HeLa and Raw264.7 cells and suppresses fatty acid oxidation. Regarding Warburg-like remodeling, the PDH phosphorylation status was altered bidirectionally (high in hyperosmolarity and low in hypoosmolarity) to osmotic stress in isolated mitochondria, suggesting that mitochondria themselves have an acute osmo-sensing mechanism. Warburg-like remodeling is required for HeLa cells to maintain ATP levels and survive under hyperosmotic conditions. Our findings suggest that cells exhibit acute metabolic remodeling under osmotic stress via the regulation of PDH phosphorylation by direct osmosensing within mitochondria.

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Fluorescence detection of metabolic activity of the fatty acid beta oxidation pathway in living cells

Abstract: Fluorescence imaging of fatty acid beta oxidation (FAO) with a fluorescent probe metabolically degraded by sequential enzyme reactions of FAO.

Cited by 34 publications

References 22 publications

Liver lipophagy ameliorates nonalcoholic steatohepatitis through lysosomal lipid exocytosis

Liver lipophagy ameliorates nonalcoholic steatohepatitis through lysosomal lipid exocytosis

A negative-solvatochromic fluorescent probe for visualizing intracellular distributions of fatty acid metabolites

Mitochondria directly sense osmotic stress to trigger rapid metabolic remodeling via PDH regulation

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