Measurement of Fatty Acid β-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Abstract

Fatty acid β-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional processes, such as ketogenesis and gluconeogenesis, which are essential to maintain whole-body glucose homeostasis and support extra-hepatic organ function in the fasted state. Fatty acid β-oxidation occurs within the mitochondria and peroxisomes and is regulated through multiple mechanisms, including the uptake and activation of fatty acids, enzyme expression levels, and availability of cofactors such as coenzyme A and NAD⁺. In assays that measure fatty acid β-oxidation in liver homogenates, cell lysis and the common addition of supraphysiological levels of cofactors mask the effects of these regulatory mechanisms. Furthermore, the integrity of the organelles in the homogenates is hard to control and can vary significantly between preparations. The measurement of fatty acid β-oxidation in intact primary hepatocytes overcomes the above pitfalls. This protocol describes a method for the measurement of fatty acid β-oxidation in a suspension of freshly isolated primary mouse hepatocytes incubated with ¹⁴C-labeled palmitic acid. By avoiding hours to days of culture, this method has the advantage of better preserving the protein expression levels and metabolic pathway activity of the original liver, including the activation of fatty acid β-oxidation observed in hepatocytes isolated from fasted mice compared to fed mice.

Figure 1.. Perfusion apparatus and perfused liver.

A) Peristaltic pump with outlet line connected to the needle used to cannulate and perfuse the liver. B) A successful cannulation is indicated by immediate and homogeneous blanching of the liver.

Figure 2.. Viability and yield of hepatocytes isolated using the procedure described herein.

Hepatocytes were isolated from male mice fed ad libitum or fasted overnight for 16–18 h. A) Hepatocyte viability and B) yield per liver. Data are reported as the mean (bars) of measurements on individual hepatocyte preparations (circles) ± SEM. Hepatocytes isolated from fed and fasted mice were compared using an unpaired two-tailed Student’s t-test.*, p<0.05.

Figure 3.. Fatty acid β-oxidation capacity in hepatocytes isolated from fed and fasted male mice and assayed in suspension.

Freshly isolated hepatocytes were pre-incubated with etomoxir (45 μM, +Eto) or DMSO (vehicle, -Eto) before the addition of the substrate mix. A) Total CPM introduced in each assay and recovered in the ASM fraction of reactions set up to estimate the background radioactivity, total (-Eto), peroxisomal (+Eto), and mitochondrial fatty acid β-oxidation. These data are shown before any correction (for background, cell number, or protein levels) or any other calculations were applied. B) Data in A) corrected for the background, total volume of the assay, normalized to 1 million viable cells, and expressed as the rate at which palmitic acid is oxidized in hepatocytes isolated from fed and fasted mice. C) Total protein corresponding to the estimated 750,000 hepatocytes/assay used. D) Data in A) corrected as in B) but normalized to mg of protein. Data are reported as the mean (bars) of measurements on individual hepatocyte preparations (circles) ± SEM. Hepatocytes isolated from fed and fasted mice were compared using an unpaired two-tailed Student’s t-test. *, p<0.05; **, p<0.01.