Activity of carnitine palmitoyltransferase in mitochondrial outer membranes and peroxisomes in digitonin-permeabilized hepatocytes. Selective modulation of mitochondrial enzyme activity by okadaic acid

Abstract

A procedure is described for the rapid measurement of the activity of mitochondrial-outer-membrane carnitine palmitoyltransferase (CPTo) and peroxisomal carnitine palmitoyltransferase (CPTp) in digitonin-permeabilized hepatocytes. CPTo activity was determined as the tetradecylglycidate (TDGA)-sensitive malonyl-CoA-sensitive CPT activity, whereas CPTp activity was monitored as the TDGA-insensitive malonyl-CoA-sensitive CPT activity. Under these experimental conditions, the respective contributions of CPTo and CPTp to total hepatocellular malonyl-CoA-sensitive CPT activity were 74.6 and 25.4%, which correlated well with the values of 76.9 and 23.1% for the respective contributions of the mitochondrial and the peroxisomal compartment to total hepatocellular palmitate oxidation. The sensitivity of CPTo to inhibition by malonyl-CoA was very similar to that of CPTp; thus 50% inhibition of CPTo and CPTp activities was achieved with malonyl-CoA concentrations of 2.6 +/- 0.5 and 3.0 +/- 0.4 microM respectively. Short-term incubation of hepatocytes with the phosphatase inhibitor okadaic acid (i) increased the activity of CPTo and the rate of mitochondrial palmitate oxidation, (ii) decreased the affinity of CPTo for palmitoyl-CoA substrate, and (iii) decreased the sensitivity of CPTo to inhibition by malonyl-CoA. By contrast, neither the properties of CPTp nor the rate of peroxisomal palmitate oxidation were changed upon incubation of cells with okadaic acid. Results indicate therefore that CPTo, but not CPTp, may be regulated by a mechanism of phosphorylation/dephosphorylation. The physiological relevance of these findings is discussed.

Carnitine palmitoyltransferase I control of acetogenesis, the major pathway of fatty acid β-oxidation in liver of neonatal swine

“…The kinetic assay for substrate (palmitoyl-CoA) yielded K m estimates ( Table 2) that were relatively high (80 -300 M), attributable in part to the nonlinear computational method and the fixed BSA concentration employed in our analysis. Notably, the increase in CPT I activity in 24-h-fed pigs was accompanied with an increase in K m for palmitoyl-CoA, illustrating that the increase in activity is related to a decrease in affinity of the enzyme for substrate, which was observed also in digitonin-permeabilized rat hepatocytes (12). The decrease would allow the enzyme to deal with the high fatty acid concentrations, as typically observed in ketotic states (11,24).…”

Leptin Induces Mitochondrial Superoxide Production and Monocyte Chemoattractant Protein-1 Expression in Aortic Endothelial Cells by Increasing Fatty Acid Oxidation via Protein Kinase A

“…Assay for CPT-1 Activity-Cells were preincubated with or without TDGA, a specific irreversible inhibitor of CPT-1, for 45 min (20). Then the medium was aspirated, and cells were washed twice with PBS.…”

Oleoylethanolamide Stimulates Lipolysis by Activating the Nuclear Receptor Peroxisome Proliferator-activated Receptor α (PPAR-α)

“…In soleus muscle strips and heart myocytes, fatty acid oxidation to CO 2 was determined by trapping released [ 14 C]CO 2 as bicarbonate in wells containing filter paper soaked with benzethonium hydroxide (1 M in methanol) (22). In hepatocytes, [ 14 C]ketone bodies, which constitute about 90% of total fatty acid oxidation products, were determined as non-volatile acid-soluble products (23).…”