In situ assay of fatty acid β-oxidation by metabolite profiling following permeabilization of cell membranes

Abstract

The mitochondrial FA ␤ -oxidation (FAO) pathway is a major contributor to cellular energy production and homeostasis, particularly once glycogen stores are depleted owing to fasting ( 1 ). Substantial amounts of energy are provided by FAO for heart, skeletal muscle, and liver function. In monogenetic defi ciencies of FAO or the carnitine shuttle system, accumulation of acyl-CoA intermediates and depletion of energy and free carnitine occur, specifically at times of increased energy demands, including fever and impaired food supply ( 2 ). Altered FA catabolism is considered key in the development of obesity-induced insulin resistance, because cellular overload with long-chain FAs is thought to exert a disastrous effect on mitochondrial function ( 3, 4 ). Cytoprotective mechanisms against FA lipotoxicity include channelling of FAs into triglyceride pools and enhancement of FA degradation via mitochondrial ␤ -oxidation ( 4 ).Thus, quantitative analysis of FAO has become increasingly important in assessing lipid-induced metabolic impairment and evaluating potential pharmacologic strategies designed to improve ␤ -oxidation fl ux ( 4 ). The most commonly applied method to assess functional FAO in cellular models has been the analysis of total FAO fl ux following incubation of intact cells with 14 C-or 3 H-labeled

Effect of Toxicants on Fatty Acid Metabolism in HepG2 Cells

“…Similar to palmitate metabolism, also the investigation of the acylcarnitine pattern in the supernatant yielded the expected results for the three established inhibitors. Based on the experience with this assay in patients with fatty acid oxidation disorders ( Ensenauer et al, 2012 ), this result could be expected. The CPT1 inhibitor etomoxir decreased the C8 to C16 acylcarnitine concentrations due to a block of the transport of palmitate into the mitochondrial matrix ( Lilly et al, 1992 ).…”

Tumor suppressor RARRES1- A novel regulator of fatty acid metabolism in epithelial cells

“…Acylcarnitines are involved in the first committed step of FAO. Long chain fatty acids are catabolized to acetyl- coenzyme A, a major fuel for mitochondrial respiration (especially during starvation), acyl-CoAs must then be converted to acylcarnitine to cross the outer mitochondrial membrane [49]. Disorders of fatty acid metabolism are typically associated with primary and secondary forms of carnitine deficiency [50].…”

Characterizing acyl-carnitine biosignatures for schizophrenia: a longitudinal pre- and post-treatment study

“…Analytical reference standards of 29 acyl-carnitines (Sigma-Aldrich, Co., St. Louis, MO, USA) were employed for quality control of compounds in samples. We defined acyl-carnitines and major acyl-carnitine fractions as free-, short-, medium-, or long-chain acyl-carnitines according to the length of carbon chains in the molecular structure (i.e., free carnitine: C0, short-chain: C2-C5, medium-chain: C6-C12, long-chain: C14-C18) 29,30 . Deuterium-labeled carnitine and acyl-carnitines (NSK-B Set, Cambridge Isotope Laboratories, Inc., Tewksbury, MA, USA) were used as internal standards, including 2 H 9 -carnitine (free carnitine, CN), 2 H 3 -acetylcarnitine (C2), 2 H 3 -propionylcarnitine (C3), 2 H 3 -butyrylcarnitine (C4), 2 H 9 -isovalerylcarnitine (C5), 2 H 3 -octanoylcarnitine (C8), 2 H 9 -myristoylcarnitine (C14), and 2 H 3 -palmitoylcarnitine (C16).…”