Interrelations between C4 ketogenesis, C5 ketogenesis, and anaplerosis in the perfused rat liver

Abstract

We investigated the interrelations between C(4) ketogenesis (production of beta-hydroxybutyrate + acetoacetate), C(5) ketogenesis (production of beta-hydroxypentanoate + beta-ketopentanoate), and anaplerosis in isolated rat livers perfused with (13)C-labeled octanoate, heptanoate, or propionate. Mass isotopomer analysis of C(4) and C(5) ketone bodies and of related acyl-CoA esters reveal that C(4) and C(5) ketogenesis share the same pool of acetyl-CoA. Although the uptake of octanoate and heptanoate by the liver are similar, the rate of C(5) ketogenesis from heptanoate is much lower than the rate of C(4) ketogenesis from octanoate. This results from the channeling of the propionyl moiety of heptanoate into anaplerosis of the citric acid cycle. C(5) ketogenesis from propionate is virtually nil because acetoacyl-CoA thiolase does not favor the formation of beta-ketopentanoyl-CoA from propionyl-CoA and acetyl-CoA. Anaplerosis and gluconeogenesis from heptanoate are inhibited by octanoate. The data have implications for the design of diets for the treatment of long chain fatty acid oxidation disorders, such as the triheptanoin-based diet.

Figures

FIGURE 1.

Scheme of C4 ketogenesis and C5 ketogenesis in the liver. Numbers refer to the following enzymes: 3-ketoacyl-CoA thiolase (1), HMG-CoA synthase (2), HMG-CoA lyase (3), and β-hydroxybutyrate dehydrogenase (4). The figure also shows the link between propionyl-CoA and the CAC via anaplerosis.

FIGURE 2.

Comparison between the uptake of octanoate (A), heptanoate (B), or propionate (C) and the production of C4 ketone bodies (β-hydroxybutyrate + acetoacetate) and C4 ketone bodies (β-hydroxypentanoate + β-ketopentanoate). ●, fatty acid uptake; ■, C4 ketogenesis; ▴, C5 ketogenesis). A, no C5 ketone bodies were detected in the presence of octanoate. A, B, C, n = 6 at zero concentration of influent fatty acid; n = 1 for the other concentrations. All liver perfusions reported in this manuscript were conducted for 20 min. All rates of substrate uptake and production (Figs. 2–5) were assayed on samples of influent and effluent perfusate taken at 14 and 18.5 min (collecting the samples over 1 min). All reported values are the means of the 14- and 18.5-min measurements.

FIGURE 3.

Competition between octanoate and heptanoate for uptake by perfused rat livers. A, constant 1 mm octanoate + increasing heptanoate concentration in influent perfusate. B, constant 1 mm heptanoate + increasing octanoate concentration in influent perfusate. The vertical scale shows the uptake of octanoate (▴) and heptanoate (▵).

FIGURE 4.

Competition between C4 ketogenesis from octanoate and C5 ketogenesis from heptanoate in perfused rat livers. A, constant 1 mm octanoate + increasing heptanoate concentration in influent perfusate. B, constant 1 mm heptanoate + increasing octanoate concentration in influent perfusate. The vertical scale shows the production of C4 ketone bodies (▴) and C5 ketone bodies (▵).

FIGURE 5.

Profile of concentrations of octanoate (●) and propionate (▴) in the effluent perfusate. The data refer to perfusions with increasing concentrations of a single fatty acid and are plotted as a function of the influent concentration of each fatty acid. The dotted line is the theoretical identity of influent and effluent concentrations.

FIGURE 6.

Labeling pattern of effluent BHB and tissue acetyl-CoA from livers perfused with increasing concentrations of [1-13C]octanoate (A) or [8-13C]octanoate (B). The figures show the molar percent enrichments (MPE) of the M1 (■) and M2 (□) mass isotopomers of BHB, the MPE of the C-1 + 2 (▴) and C-3 + 4 (▵) acetyls of BHB, and the MPE of liver acetyl-CoA (●). The labeling patterns of ketone bodies (Figs. 6 and 7) were assayed in samples of effluent perfusate taken at 14 and 18.5 min (collecting the samples over 1 min). All reported values are the means of the 14 and 18.5 min measurements.

FIGURE 7.

Sharing of acetyl groups between C4 and C5 ketogenesis reflected by the mass isotopomer distribution of BHB and BHP. A, labeling pattern of BHB in perfusions with constant 1 mm [1-13C]heptanoate + increasing concentrations of unlabeled octanoate. B, labeling pattern of BHP in perfusions with constant [5,6,7-13C3]heptanoate and increasing concentrations of [1-13C]octanoate. C, labeling pattern of BHP in perfusions with constant [5,6,7-13C3]heptanoate and increasing concentrations of [8-13C]octanoate. D, labeling pattern of BHP in perfusions with constant [1-13C]octanoate and increasing concentrations of unlabeled heptanoate.

FIGURE 8.

Mass isotopomer distribution of HMG-CoA (A) and HEG-CoA (B) in livers perfused with constant 1 mm [1-13C]heptanoate and increasing concentrations of unlabeled octanoate.

FIGURE 9.

Mass isotopomer distribution of BHB-CoA and AcAc-CoA in livers perfused with increasing concentrations of [1-13C]octanoate.

FIGURE 10.

Anaplerosis and glucose labeling from increasing concentrations of [13C3]propionate (♦) or [5,6,7-13C3]heptanoate (■, ▴). The perfusions with increasing [5,6,7-13C3]heptanoate concentrations were conducted in the absence (▴) or presence (■) of constant 1 mm [1-13C]octanoate. A, relative anaplerosis expressed as the m3 enrichment ratio (succinyl-CoA)/(propionyl-CoA). B, absolute anaplerosis. C, M2 + M3 labeling of glucose in the effluent perfusate.