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2
*
Service de Nutrition et Diabétologie, Hôpital Haut-Lévêque, F-33600 Pessac France and
Centre de Résonance Magnétique des Systèmes Biologiques, UMR 5536 CNRS-UB2, F-33076 Bordeaux Cedex France
2To whom correspondence and reprint requests should be addressed. E-mail: mcdb{at}rmsb.u-bordeaux2.fr.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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20 min,
dependent on the chain length and concentration of the fatty acid (FA).
The initial Vi was unchanged for acetate and
the ATP level stabilized at 58% of the initial level. Both butyrate
and octanoate induced a dose-dependent increase in
Vi. This may reflect an ATP-consuming
process for the intracellular pH regulation observed during the
acidosis associated with the ß-oxidation pathway. At the new steady
state, the ATP concentration was 
45% of the initial level for both
FA. VO2 was both rapidly and reversibly increased, and the
change was a function of butyrate or octanoate concentration and of the
chain length. Km values were similar for
butyrate and octanoate. Because all of the effects were similar for
butyrate and octanoate, in contrast to acetate, we suggest that the
impairment of the energy metabolism by butyrate resulted from an
increase in the FADH2/NADH ratio due to ß-oxidation. In
conclusion, the difference in the hepatic oxidation pathways of two
products of intestinal fermentation (acetate and butyrate) explains
their different actions on energy metabolism.
KEY WORDS: • short-chain fatty acids • butyrate • nuclear magnetic resonance • respiration • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Three SCFA account for 83% of all SCFA produced, and
these are distributed in a fairly constant ratio of 60:25:15
(acetate/propionate/n-butyrate) (2)
. It has
been proposed that they be used to supplement total parenteral
nutrition solutions and hence inhibit the small-bowel mucosal
atrophy that would otherwise occur when luminal nutrients are absent
(3
,4)
. SCFA also contribute to energy requirements, 5–9%
in both humans and rats (5
,6)
. Because an increase in the
amount of fiber in the diet will undoubtedly increase the amount of
SCFA produced, the contribution of SCFA to the energy supply of the
body may increase considerably as the amount of dietary fiber
increases.
Moreover, SCFA may indirectly influence carbohydrate and lipid
metabolism (7
,8)
. It has been suggested (9)
that some of the beneficial effects of high carbohydrate, high fiber
diets on carbohydrate and lipid metabolism are mediated by the
metabolism of SCFA in the liver. SCFA, which are normally produced in
the colon, are readily absorbed by the colonic mucosa and metabolized
by both the colonic mucosa and the liver (10)
. Hepatic
metabolism and clearance of SCFA are substantial because portal
concentrations are 150% greater than those simultaneously determined
in the hepatic vein and 375% greater than systemic concentrations
(11)
.
Without consideration of the chain length, several aspects of fatty
acid (FA) metabolism in the liver have been described, especially the
stimulation of ATP-consuming pathways, such as gluconeogenesis,
ketogenesis, triglyceride synthesis and secretary processes
(12
,13)
. ATP use is also enhanced when FA are activated by
conversion to CoA derivatives (14)
. Under physiologic
conditions, ATP consumption must be offset by increased ATP synthesis
because the energy steady state of the cell must be maintained. The
reduced compounds (NADH + H+,
FADH2) resulting from ß-oxidation of FA as
fuels are reoxidized in the mitochondrial respiratory chain and are
thought to lead in part to ATP generation. For a given FA
concentration, the energy supply is directly dependent on the chain
length. However, medium- or long-chain FA have been demonstrated to
impair mitochondrial activity, and their effector roles could be linked
to a mechanism similar to the uncoupling of oxidative phosphorylation
in mitochondria (15
16
17)
.
To our knowledge, no study of the time course of the effect of SCFA on hepatic energy metabolism (nucleotide content and respiratory activity) has been reported. In addition to measuring the variation in O2 consumption, our aim was to use nuclear magnetic resonance (NMR) to continuously measure changes in the energy metabolism of intact livers. The organ isolated from fed rats was perfused, for prolonged periods of time, with different concentrations of even-chain SCFA, namely, acetate and butyrate. The results are compared with the data obtained with octanoate as substrate to determine the influence of the chain length.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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High grade chemicals were purchased from Sigma Chemical (St. Louis, MO)
except where otherwise specified. Media were diluted daily from
concentrated stock solutions. Standard Krebs-Heinseleit buffer
(KHB) was composed of (mmol/L) 120 NaCl, 4.70 KCl, 1.20
MgSO4, 25 NaHCO3, 1.20
KH2PO4-K2HPO4, 1.30
CaCl2, 0.30 Na-pyruvate and 2.10 Na-lactate (pH
= 7.35 at 37°C). Substrates (acetate, butyrate and octanoate)
were added to the KHB as sodium salts. In humans, dietary production of
SCFA in the colon and their hepatic levels have been reported to be
200–800 mmol/d (1
,18)
and 148 ± 42
µmol/L (11)
, respectively. According to
these physiologic reports and because octanoate is water insoluble up
to 4.7 mmol/L, we chose to add acetate and butyrate in the 0.05–5
mmol/L range and octanoate in the 0.05–3 mmol/L range. Only
even-chain substrates were selected to allow comparisons in their
metabolism.
Male Wistar rats (Center d’élevage Depré, St Doulchard,
France) weighing 80–120 g consumed food ad libitum. The diet
composition is specified in Table 1
. Rats were anesthetized by intraperitoneal injection of pentobarbital
sodium (50 mg/kg); liver perfusion through the portal vein (antegrade
perfusion technique) was described previously (19)
.
Perfusion with nonrecirculating medium was chosen to avoid
recirculation of hepatic catabolites, and the bile duct was cannulated
to avoid mixing of bile with the liver effluent. Briefly, the liver
(4–6 g) was perfused with KHB at 37°C. The temperature of the
perfusion circuit was regulated with a thermostatic bath. The perfused
liver was then excised from the rat abdomen and transferred into a 20
mm-diameter NMR cell. The beginning of the liver perfusion always
occurred between 1300 and 1400 h to minimize the effects of
differences in dietary fiber fermentation and endogenous SCFA
production. All experiments were conducted after a 30-min metabolic
equilibration period.
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The sequence protocol at 37°C was as follows: 1) initial
KHB perfusion (30 min); 2) KHB + substrate perfusion (70–90
min); and 3) rinse with KHB perfusion (30 min). A control
group consisted of livers (n = 3) perfused only with
KHB for 100 min. The sequence protocol was applied to three groups of
livers devoted to different measurements. The first group was subjected
to continuous NMR measurements to follow ATP levels and to determine
glycogen content. The second group was devoted to respiratory
measurements. The third group corresponded to experiments with
sequential NMR and respiratory measurements (requiring the removal of
the NMR cell from the magnet). For each group, n = 2–9, as specified in the results section, for each substrate at each
concentration. This study complied with NIH guidelines
(20)
.
Liver oxygen consumption measurements.
NMR experiments require a long thermostatically controlled perfusion and suction lines, which could affect oxygen consumption measurements. As a consequence, additional experiments were performed outside the magnet, with the isolated perfused liver placed into a NMR cell similar to that used for the NMR experiments. Respiration of the liver was assessed using O2 Clark electrodes, placed in the perfusion circuit just before and after the liver, in conjunction with continuous monitoring using an oxygen meter (model 781; Strathkelvin Instrument, Glasgow, Scotland). Oxygen consumption (VO2) [µmol O2/(min · g wet liver)] = (perfusate O2 content - effluent O2 content) x liver flow rate [mL/(min · g wet liver)] where O2 concentration was expressed in mmol/L.
31P NMR measurements.
NMR measurements on isolated liver were performed on a Bruker DPX-400 wide-bore spectrometer (Wissembourg) operating at 9.4 T. 31P and 13C NMR spectra were recorded at 161.9 and 100.6 MHz, respectively, with a 31P/13C double-tuned 20-mm probe. The magnetic field was adjusted on the water proton signal. 31P NMR spectra were obtained every 2 min [240 free induction decays (FID)] without proton decoupling. Radiofrequency pulses of 25 µs duration (70° flip angle) with 0.2 s acquisition time, 0.5 s total recycling time, 10,000 Hz spectral width and 4K data points were used for data acquisition. 13C NMR spectra were proton-decoupled using a gated bilevel mode; a high level of 1H irradiation was used during acquisition and a low level during relaxation delay. Each 13C NMR spectrum corresponded to the Fourier transformation of 500 FID, resulting from a 66° radiofrequency pulse repeated every second (25,000 Hz spectral width). A Lorentzian line broadening of 15 Hz was applied before Fourier transformation for both 31P and 13C NMR spectra.
Chemical shift of phosphorylated metabolites was expressed relative to the position of resonance in the frequency scale (chemical shift in ppm) of an external reference of 13 µmol methylene diphosphonic acid (set at 18.40 ppm) contained in a sealed capillary and glycerophosphorylcholine as an internal standard set at 0.47 ppm. 13C chemical shifts were expressed from an external silicone reference (at 1.45 ppm).
ATP represented at least 80% of ß-nucleoside-5'-triphosphate
(ßNTP) (21)
. Hepatic ATP levels were estimated
from peak areas and expressed as a percentage of the initial value
obtained at the end of the 30-min equilibration period in KHB. During
this period, any livers showing an increase in the intensity of
inorganic phosphate (Pi) resonance occurring with a concomitant
decrease in NTP signals (probably reflecting some partial lobe
ischemia) were discarded.
Changes in intracellular pH (pHi) were determined from the chemical
shift of the intracellular Pi signal using a titration curve
constructed at 37°C according to the Henderson-Hasselbach
equation (19)
.
Evaluation of the apparent rate of ATP depletion.
Liver ATP content was decreased to varying degrees under the various experimental conditions. Under our experimental conditions, the time course of ATP concentration change during the first 20 min of the substrate perfusion could be fitted to a single exponential (validated by a r2 value >0.91; P = 0.05) of the form: ATP (t) = A exp(-kt), where k is a time constant and A the liver ATP concentration at the onset of the substrate perfusion. To compare all experimental groups, the values at the onset of ATP depletion were arbitrarily fixed to 100%. The derivative of the equation provides the apparent velocity of the ATP decrease: V(t) = d(ATP(t))/dt. The value of V(t) for t = 0 gave the initial rate of net ATP consumption (Vi).
Biochemical assays.
Liver effluents (3 mL) were collected to determine glucose and lactate excretions enzymatically at each phase of the protocol (glucose oxidase and lactate dehydrogenase; Sigma Diagnostics). The collection of samples was systematically performed synchronously with the 13C NMR acquisitions. Glucose and lactate excretions were expressed in µmol/(min · g wet liver).
Statistical procedures.
All results were expressed as means ± SEM. Statistical analysis was performed with the software package Statworks (Cricket Software, London, UK). One-way ANOVA was carried out to test for any differences among the mean values; the analyzed factor was the substrate concentration or the nature of the substrate. Two-way ANOVA was performed for the initial rate of liver ATP consumption Vi and for the hepatic O2 consumption VO2; the analyzed factors were the nature and the concentration of substrates. A t test was used after ANOVA to identify significant differences between different substrates or between different concentrations. Differences with a P-value < 0.05 were considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Typical 31P NMR spectra of an isolated rat liver
perfused at 37°C with KHB are shown in Figure 1A
and
C
. The liver ATP concentration (2.60 ± 0.05 µmol/g wet
liver; n = 16) determined from the ßNTP resonance
area was quite stable during the initial 30-min period of KHB perfusion
in all groups and throughout the complementary perfusion in the control
KHB group. A slow ATP degradation [9.01 ± 0.68 nmol/(min · g
wet liver; n = 56) during the initial 30-min period]
was observed, leading to an ATP level of 65% of the initial content
after a total of 100 min KHB perfusion (Fig. 1B
).
|
), acetate or octanoate
after the 30-min initial KHB perfusion enhanced the initial rate of ATP
net consumption (Vi) compared with KHB
(Table 2
, P < 0.0001). Figure 2
shows typical experiments performed in the presence of 1.5 mmol/L of
each substrate. This increase of the
Vi was transient, and a new steady
state of ATP concentration was observed independent of the species and
concentration of the substrates used. For a given substrate (butyrate
or octanoate), the time of the onset of this steady state was
significantly shorter when its concentration was higher (Fig. 3
). For a given concentration of the substrates, the time of the onset
was lower when the length of the chain was longer (Fig. 3)
. The ATP
concentration stabilized at 41 ± 3% (n = 19)
and 47 ± 3% (n = 13) of the initial ATP level in
the presence of butyrate and octanoate at concentrations 1.5 mmol/L,
respectively, and at 58 ± 3%
(n = 21) in the presence of acetate, independent of its
concentration (P < 0.0001 vs. butyrate, P
< 0.01 vs. octanoate). The new steady state was characterized by
an ATP degradation near that of the KHB control. A subsequent rinse
period using KHB did not change the ATP concentration obtained in the
presence of substrates.
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and the
half-maximal effect (Km) was at
0.7–1.0 mmol/L. The Vm of this ATP
net consumption was 55–65% higher in the presence of octanoate
(n = 6) than in the presence of butyrate (n
= 7; P < 0.001 at 1.5 mmol/L), suggesting that it
was dependent on the carbon number. Thus, in terms of a concentration
corresponding to an equivalent number of carbons,
Vi was similar for octanoate at 0.5
mmol/L and butyrate at 1.0 mmol/L. Changes in intracellular pH.
The pHi value was 7.20 ± 0.06 during KHB perfusion. The addition
of acetate did not affect pHi at any concentration. A rapid and sharp
decrease in the pHi was observed in the presence of butyrate
(Fig. 4
). The pHi decrease was significant at 0.5 mmol/L butyrate and was
dose dependent (linear correlation, r2
= 0.98) up to 10 mmol/L (-0.40 pH units), the highest dose used. The
pHi returned to its basal value with a mean delay of 20 min except for
the highest concentrations for which the new steady-state pHi was
more acidotic (7.08 ± 0.09 at 10 mmol/L). In the presence of
octanoate, a similar dose-dependent decrease in pHi was observed in
the range of concentrations used (data not shown).
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Under initial KHB perfusion, the O2 liver
consumption was 2.45 ± 0.15 µmol
O2/(min · g wet tissue). The addition of the
substrates increased the respiration rate (VO2).
The delay in reaching the new steady state was <20 min. The increase
in VO2 was dependent on the butyrate and
octanoate concentrations in the range of doses tested (Fig. 5
) with the half-maximal effect at 100 µmol/L. With
acetate, VO2 was maximum for concentrations as
low as 10 µmol/L. Thus, for each substrate, 1 mmol/L was
the saturating concentration giving the maximum
O2 consumption. The increase of
VO2 was also dependent on the chain length
(Fig. 6
); the increase in VO2 at the steady state was
fivefold higher (n = 3) in the presence of butyrate
than in presence of acetate and 60% higher (n
= 3) in the presence of octanoate than in the presence of butyrate
(Fig. 5)
. Whatever the nature and the concentration of the substrate,
its effect on the O2 consumption was reversible
during the KHB rinse.
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Typical 13C NMR spectra of isolated rat livers
during the 30-min control perfusion in KHB at 37°C are shown in
Figure 7A
and C
. Glycogen was characterized mainly by a narrow signal at 100.5 ppm
(glycogen C-1 resonance). Glycogen signals also contributed to
resonances between 60 and 100 ppm. In the absence of carbohydrates in
the perfusate, glycogenolysis occurred in the KHB group, and its
apparent rate, evaluated by the changes of the area of glycogen C-1
resonance from consecutive spectra throughout the perfusion of the same
liver, was 0.92 ± 0.08%/min (100% is considered to be the
initial content); only 10% was detectable after 100 min perfusion
(Fig. 7B
). The presence of acetate, butyrate and octanoate
induced lower decreases in the intensity of the
13C resonances of glycogen, which were detectable
in the presence of KHB after 100 min of perfusion. After 120 min of
substrate perfusions, the residual glycogen was indeed near 30–40% in
the presence of acetate (Fig. 7D
), butyrate and octanoate,
whatever the concentrations.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,22)
.
Moreover, the use of SCFA to supplement parenteral nutrition of
individuals with short bowel syndrome or intestinal malabsorption
syndromes may improve some intestinal functions (4
,23)
.
Acetate, butyrate and propionate constitute the majority of products
derived from intestinal fermentation. After their uptake by the colon,
they are metabolized in part by the colonocytes, and the remaining
fraction reaches the liver via the portal vein. Any evaluation of the
nutritional effect of SCFA has to include the study of the relationship
between hepatic energy metabolism and their oxidation pathways.
The present work on the intact liver used 31P NMR
to study continuously the evolution of the amount of ATP and hence the
evolution of energy metabolism in the presence of SCFA perfused under
physiologic concentrations. Respiratory activity was determined
simultaneously using Clark electrodes. One of the reasons for using
isolated perfused livers was to evaluate the roles of SCFA in the
absence of effects originating from their uptake and metabolism by
colonocytes (11)
. Moreover, to be free from the known
reciprocal influences of SCFA on their absorption and/or their liver
metabolism (24
,25)
, our study concerned only the inherent
effect of each substrate. We intentionally selected substrates with
even-chain (acetate, butyrate and a medium-chain FA,
octanoate), which lead to the production of the same metabolite, i.e.,
acetyl-CoA. Indeed the odd-chain FA, propionate, takes a
different enzymatic pathway, leading to succinyl-CoA, which in turn
bypasses a large part of the citric acid cycle and is a
precursor of gluconeogenesis. The production of acyl-CoA via
short-chain acyl-CoA synthase consumes 1 ATP/mol. Butyryl-CoA
and octanoyl-CoA are ß-oxidized, providing acetyl-CoA and
reduced compounds; acetyl-CoA finally enters into the citric acid
cycle. The rate of respiration depends directly on the amount of
reduced compounds (FADH2 and NADH +
H+) produced during either ß-oxidation or the
citric acid cycle. The reoxidation of these reduced compounds can
theoretically provide ATP to maintain tissue ATP content. In the basal
condition (KHB perfusion), we previously reported low metabolic
activity in the liver (26)
, corresponding to 10–20% of
the maximal oxidative phosphorylation capacity; moreover, the ATP
provided by glycolysis contributes 30% of the total liver ATP
synthesis (27)
. Here, in the case of the livers of fed
rats, because glycogenolysis occurs without an increase in glucose
excretion, some ATP can be provided from glycolysis until the total
hydrolysis of glycogen occurs. However, the glycogenolysis rate was
lower in presence of substrates than with KHB. These results suggest
that the substrates studied, leading to acetyl-CoA, can reduce
glycolysis and net hepatic glycogen breakdown.
The respiration rate was increased in the presence of all substrates.
To reach a new steady state of respiratory activity required 20 min of
perfusion, with this delay probably reflecting the time necessary to
obtain both extra- and intracellular equilibrium of substrate
concentration and the resulting metabolic effect. The stimulation of
respiration was dependent on butyrate and octanoate concentrations as
previously demonstrated for octanoate (14)
. However, the
respiration rate increased with chain length for a given concentration,
which can be explained by the ß-oxidation pathway (28)
as shown by the lowest respiration rate in the presence of acetate.
Because respiration was greatly stimulated in the presence of butyrate
and octanoate, an increase in coupled-ATP synthesis can be
expected. However, the ATP content (resulting theoretically from both
consumption and synthesis) was nonreversibly decreased to reach a new
steady state. A decrease of ATP content was reported previously with
octanoate and oleate (12
,29)
or with linoleate and
linolenate carried by liposomes (30)
. In the present work,
we demonstrated that a liver perfusion time of at least 20 min in the
presence of butyrate and octanoate is required to obtain a new ATP
content steady state in the isolated liver. Such a phenomenon has not
been clearly demonstrated in other studies due to the absence of
continuous measurements of ATP levels and the use of perfusion times of
either 10 (29)
or 30 min (12)
. In a model of
isolated hepatocytes, 40 min of perifusion with octanoate or oleate was
required to reach the steady-state concentrations of adenine
nucleotides (31)
.
At the onset of the substrate effect, the concentration dependence (up
to 1–2 mmol/L of substrates) of the ATP net consumption probably
represents in part the consequence of the hydrolysis of 1 mol ATP/mol
of substrate to form 1 mol acyl-CoA. However, the initial rate of
ATP net consumption was higher in the presence of butyrate and
octanoate than in the presence of acetate, whereas the acyl-CoA
activation step occurred in all cases. We postulate that a part of the
initial ATP consumption in the presence of butyrate and octanoate was
associated with pHi homeostasis. Indeed, very similar
dose-dependent decreases in pHi were observed for butyrate and
octanoate, which could be due to the following: 1) the rapid
cytosolic acid load and/or 2) the delayed increase of NADH +
H+ linked to the mitochondrial ß-oxidation
pathway. Subsequently, pHi tended to return to the basal value,
indicating a pHi regulation. The liver pHi homeostasis implies
participation of ion transport mechanisms
[Na+-H+ and
Na+-HCO3-
transporters (32)
] with indirect activation of the
Na+/K+-ATPase [known to
require 25% of the cellular ATP turnover under basal conditions
(33
,34)
]. The absence of a significant pHi decrease
during acetate perfusion at any concentration used could be due to the
rapid metabolism of this substrate (35)
and/or the absence
of ß-oxidation. Our results suggested that no SCFA-induced
decrease in hepatic pHi occurs under physiologic conditions because
their portal concentration resulting from intestinal fermentation
remains <0.5 mmol/L (11)
. However, higher liver
concentrations provided either by an excessive diet supply of dairy
products or by SCFA-supplemented nutrient solutions may impair the
pHi homeostasis.
All of the results relating to the effects of butyrate and octanoate on
the liver implied that the pathways of ATP synthesis (oxidative
phosphorylation, glycolysis) were insufficient to compensate for the
ATP consumption at the beginning of the substrate perfusion. For the
same range of substrate concentrations, the lowest respiration rate
(associated with the least amount of reduced compounds generated) and
the highest final ATP concentration were observed with acetate. Because
the well-known inhibitory effect of acetyl-CoA on glycolysis
(and its associated ATP synthesis) is the same for all FA, the latter
results suggest that a more efficient coupling between respiration and
ATP synthesis occurs in the presence of acetate compared with butyrate
and octanoate. Octanoate and longer-chain FA have been demonstrated
to impair mitochondrial activity as the consequence of an intrinsic
uncoupling of the respiratory chain (direct effect)
(15
16
17)
or a change in the proportion of electrons
supplied to the coupling sites due to the increase of
FADH2/NADH ratio resulting from ß-oxidation
(17)
. The latter process could explain the similar
metabolic effects observed for butyrate and octanoate in contrast to
acetate. In conclusion, the difference in the hepatic oxidation
pathways of the two products of intestinal fermentation examined
(acetate and butyrate) explains their different action on energy
metabolism with butyrate being more similar in its effects to
longer-chain fatty acids.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: FA, fatty acids; FID, free induction decay; KHB, Krebs-Henseleit buffer; NMR, nuclear magnetic resonance; NTP, nucleoside-5'-triphosphate; pHi, intracellular pH; SCFA, short-chain fatty acids; Vi, initial rate of net consumption of liver ATP; Vm, maximum initial rate of net consumption of liver ATP; VO2, oxygen consumption.
Manuscript received January 2, 2001. Initial review completed February 15, 2001. Revision accepted April 20, 2001.
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NTP)
and diphosphates (ßNDP); (g) 
-NTP and 
pHi) in the
presence of butyrate for five individual typical experiments, expressed
in pH units (
