Decrease in oxidative phosphorylation yield in presence of butyrate in perfused liver isolated from fed rats

Abstract

Background: Butyrate is the main nutrient for the colonocytes but the effect of the fraction reaching the liver is not totally known. A decrease in tissue ATP content and increase in respiration was previously demonstrated when livers were perfused with short-chain fatty acids (SCFA) such as butyrate, or octanoate. In fed rats the oxidative phosphorylation yield was determined on the whole isolated liver perfused with butyrate in comparison with acetate and octoanoate (3 mmol/L). The rate of ATP synthesis was determined in the steady state by monitoring the rate of ATP loss after inhibition of (i) cytochrome oxidase (oxidative phosphorylation) with KCN (2.5 mmol/L) and (ii) glyceraldehyde 3-phosphate dehydrogenase (glycolysis) with IAA (0.5 mmol/L). The ATP flux, estimated by 31P Nuclear Magnetic Resonance, and the measured liver respiration allowed the ATP/O ratio to be determined.

Results: ATP turnover was significantly lower in the presence of butyrate (0.40 +/- 0.10 micromoles/min.g, p = 0.001, n = 7) and octanoate (0.56 +/- 0.10 micromoles/min.g, p = 0.01, n = 5) than in control (1.09 +/- 0.13 micromoles/min.g, n = 7), whereas perfusion with acetate induced no significant decrease (0.76 +/- 0.10 micromoles/min.g, n = 7). Mitochondrial oxygen consumption was unchanged in the presence of acetate (1.92 +/- 0.16 vs 1.86 +/- 0.16 for control) and significantly increased in the presence of butyrate (p = 0.02) and octanoate (p = 0.0004) (2.54 +/- 0.18 and 3.04 +/- 0.15 micromoles/min.g, respectively). The oxidative phosphorylation yield (ATP/O ratio) calculated in the whole liver was significantly lower with butyrate (0.07 +/- 0.02, p = 0.0006) and octanoate (0.09 +/- 0.02, p = 0.005) than in control (0.30 +/- 0.05), whereas there was no significant change with acetate (0.20 +/- 0.02).

Conclusion: Butyrate or octanoate decrease rather than increase the rate of ATP synthesis, resulting in a decrease in the apparent ATP/O ratio. Butyrate as a nutrient has the same effect as longer chain FA. An effect on the hepatic metabolism should be taken into account when large quantities of SCFA are directly used or obtained during therapeutic or nutritional strategies.

Figure 1

Typical ³¹P Nuclear Magnetic Resonance spectra of an isolated rat liver. A: After 30-min control KHB normothermic perfusion. Major resonances are assigned to (a) phosphomonoesters; (b) phosphocholine; (c) intracellular inorganic phosphate (Pi); (d) glycerol-3-phosphoryl ethanolamine and glycerol-3-phosphorylcholine; (e) nucleoside-5'-triphosphates (γNTP) and diphosphates (βNTP); (f) α-NTP and α-NDP; (g) nicotinamide adenine dinucleotide; (h) uridine-5'-diphosphoglucose; (i) βNTP. The reference (methylene-diphosphonic acid) is not shown (18.40 ppm) (number of scans = 148). B: 20 min after a perfusion of butyrate 3 mmol/L, effect (from the bottom to the top) of the simultaneous addition of IAA 0.5 mmol/L (2 min) and KCN 2.5 mmol/L (10 min) (number of scans = 60).

Figure 2

Time course of liver ATP content throughout the entire protocol perfusion. Simultaneous inhibition of glycolytic ATP synthesis by IAA (0.5 mmol/L, 2 min) and of ATP mitochondrial synthesis by KCN (2.5 mmol/L, 10 min) for two typical experiments performed in presence of (A) Krebs-Henseleit Buffer and (B) butyrate (3 mmol/L). The results are expressed as percent of ATP content, the 100% value being at the beginning of KHB perfusion (2.60 ± 0.05 μmol/g liver wet weight).