1 Division of Nutritional Sciences and 2 Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The current studies were performed to better
understand the physiological relevance of acetate in the poorly
ketogenic piglet and to determine if endogenous acetogenesis rises with
increased mitochondrial fatty acid -oxidation, analogous to
ketogenesis. Plasma acetate concentration values in newborn, fasted, or
suckled piglets (230-343 µM) were at least 10-fold higher than
the ketone bodies, a pattern opposite to that in 24- to 48-h suckled
rats (77-175 µM). Employing continuous infusion techniques with
sodium [3H]acetate
tracer in fasting ~40-h-old piglets, acetate rate of appearance
(Ra) was found to be 34 ± 4 µmol · min
1 · kg
body wt
1. This basal
Ra was double that observed in
animals coinfused with sodium
[1-14C]hexanoate
(P < 0.001), despite active
oxidation of the latter as determined by
14CO2
production. Active acetogenesis in vivo and relatively abundant acetate
in piglet blood are consistent with the hypothesis that acetate plays
an important physiological role in piglets. However, the negative
impact of hexanoate oxidation upon acetate
Ra and the lack of significant
changes in circulating acetate in newborn, suckled, and fasted piglets
draws into question the extent of analogy between acetogenesis and
ketogenesis in vivo.
-oxidation; newborns; ketone bodies; medium-chain fatty
acids
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE SUCKLING PERIOD, bridging intrauterine life and the nutritional independence of weaning, is characterized by a heavy reliance upon milk-derived lipids to cover the energetic and growth requirements of the neonate. The postpartum transition toward a lipid-based metabolism has been best characterized in the rat, a species in which birth triggers mechanisms working in concert to support fatty acid oxidation and ketogenesis (reviewed in Ref. 10). In particular, carnitine palmitoyltransferase I (CPT-I, EC 2.3.1.21) and mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase (HMG-CoA synthase, EC 4.1.3.5), enzymes central to ketogenesis, are upregulated in newborn suckled rat liver (10, 32) and small intestine (11). The dramatic fall of insulin coupled to rising glucagon and nonesterified fatty acids (NEFA) in the circulation of rat pups after birth (10) appears to spur gene expression for these enzymes, as the genes encoding these proteins are sensitive to the prevailing hormonal and fatty acid milieu (5, 11, 32).
The extent to which this model applies across mammalian taxa is not
firmly established. The suckling piglet, for example, does not develop
the neonatal ketonemia evident in some species (2, 27) and may not
display a postpartum pancreatic hormone shift equivalent to the rat
(27, 37). The piglet's negligible capacity for hepatic ketogenesis (1,
2, 8, 20, 24, 28) could be the result of unique differences in liver
activities of mitochondrial HMG-CoA synthase and/or CPT-I, the
former being negligible around birth (3, 8) and the latter being
particularly sensitive to malonyl-CoA inhibition (8).
Interestingly, recent characterization of the end products derived
from medium- (MCFA) or long (LCFA)-chain fatty acid -oxidation in
piglet liver revealed that acetogenesis far surpasses ketogenesis
in vitro (1, 20, 24), a pattern opposite that observed in rats (1, 35). However, the physiological relevance of acetogenesis vs. ketogenesis during the neonatal period of piglets could not be determined from
those studies. Furthermore, despite evidence of in vitro fatty acid
carbon flux to acetate in the liver of piglets (1, 20, 24) and rats
(19, 25, 34, 35), the prospect that mitochondrial acetogenesis is
analogous to ketogenesis has not been fully explored.
From a physiological standpoint, active acetogenesis and low
ketogenesis in vitro suggest that acetate turnover more profoundly impacts piglet metabolism than do the ketone bodies. If true, this
scenario would differ substantially from models in which ketone bodies
serve as important glucose-sparing fuels and potential brain lipid
precursors during neonatal development (see Ref. 10). The current
studies were designed to gain insight into the physiological roles of
acetate in the newborn piglet model and to explore the possibility that
mitochondrial acetogenesis (like hepatic mitochondrial ketogenesis in
rats) increases concomitant with elevated mitochondrial -oxidation
in vivo. As a first approach, plasma concentrations of acetate and
ketone bodies were compared during the first 48 h of life in piglets.
Circulating acetate levels were also determined in neonatal rats,
animals with substantial ketogenic capacity during the neonatal period
(10). A second set of experiments measured the kinetics of endogenous
(nonfermentative) acetate in piglets determined in the absence or
presence of the mitochondrial fuel hexanoate to measure basal acetate
rate of appearance (Ra) and the
impact of mitochondrial
-oxidation upon
Ra, respectively. Results showing
comparatively elevated plasma acetate and a basal acetate
Ra reflecting substantial
production/utilization are consistent with an important physiological
role for acetate vs. ketone bodies in piglets. However, some aspects of
acetate metabolism in piglets draw into question the extent of analogy
between acetogenesis and ketogenesis under the current experimental
conditions.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Neonatal Changes in Circulating Acetate and Ketone Bodies
Animals and blood sampling. All work was approved by the University of Illinois Laboratory Animal Care Advisory Committee. For the study of changes in acetate and ketone body concentrations over the first 2 days postpartum in swine, commercial crossbred piglets (birthweight 1.85 ± 0.07 kg) were identified from attended farrowings and assigned randomly to the following groups: newborn unsuckled (0.5-2 h old), 24 h old unsuckled, 24 h old suckled, and 48 h old suckled. Animals left unsuckled 24 h were confined to farrowing crates with access to a heat lamp. At the respective time points, blood was sampled by jugular venipuncture into heparinized Vacutainer tubes (Becton-Dickinson, Rutherford, NJ). Blood was placed in ice until centrifugation at 5,000 g (4°C) to obtain plasma, which was stored atAnalysis of volatile fatty acids and ketone bodies. Samples were analyzed using a slight modification of the gas-liquid chromatography (GLC) method of Rémésy and Demigné (33). To precipitate protein and extract volatile fatty acids (VFA), ~2-3 vol of ice-cold 100% ethanol were added to samples (450-700 µl) combined with 0.1 µmol of isobutyric acid internal standard. After centrifugation (10 min, 13,000 g, 4°C), the supernatant fractions were transferred to microcentrifuge tubes containing 6 µmol of NaOH, yielding a final pH of ~8. Contents were dried using a Speedvac (Savant, Farmingdale, NY). Just before GLC analysis, samples were redissolved in 80 µl of ice-cold 6.25% metaphosphoric acid and clarified by centrifugation (15 min, 4,000 g, 4°C) through 0.45-µm nylon microcentrifuge tube filters (Lida Manufacturing, Kenosha, WI). Samples were transferred to 200-µl autosampler vial inserts placed in crimp-top vials and loaded into an HP 7673 autosampler attached to an HP 5890 Series II GLC instrument (Hewlett-Packard, Wilmington, DE).
Samples (2 µl) were injected into the gas chromatograph, which employed the following conditions: a Supelco (Bellefonte, PA) GP 10% SP-100/1% H3PO4 on 80/100 Chromosorb WAW column (6 ft × 4 mm ID), N2 carrier (73 ml/min), injector temperature 175°C, oven temperature 125°C, and a flame-ionization detector (FID) set at 180°C. Conversion factors (µM/FID response peak area) and assay linearity were determined each day from authentic VFA mixed standards (fatty acid chain lengths C2-C5, including isobutyric acid) covering the range of sample concentrations. Water blanks were chromatographed between each sample. No loss of acetate or isobutyrate was observed in samples through at least 12 h or in mixed standards in crimp-top vials over 5 days at room temperature (data not shown). The method separated VFA and MCFA through at least hexanoate with high resolution (not shown). The coefficient of variation of duplicate sample analyses averaged 5.4 ± 0.4%.
To assess the potential for artifactual elevation of acetate concentration due to contamination from reagents or other sources, equivalency of acetate and isobutyrate recovery was checked via the following procedures. Mixed standards were processed using the normal protocol, and recovery of individual VFAs was calculated by comparison with directly injected standards. Recovery was consistently 10% higher for acetate vs. isobutyrate, confirmed by recovery of radioactivity in a subset in which tracer sodium [1-14C]acetate (Research Products International, Mt. Prospect, IL) was added with standards before processing. Thus all reported values have been adjusted to account for this difference, with mean adjusted acetate recovery equal to 74 ± 1% over all samples (average recovery of radioactivity in 2 plasma samples spiked with sodium [1-14C]acetate was 72%). No contamination of acetate in assay or blood collection reagents was detected by GLC. Using this method of VFA analysis, plasma acetate concentration in samples obtained from overnight-fasted adult humans (71 ± 6 kg; n = 4) was consistent with the literature (73 ± 8 µM; e.g., see Ref. 31).
Plasma -hydroxybutyrate (
-OHB) concentration in piglets was
measured as described previously (2), with AcAc concentration measured
as the difference in
-OHB concentration between assays of matched
samples with or without treatment by
-OHB dehydrogenase plus NADH.
Acetate Kinetics
Animals and surgery. To facilitate measurement of acetate kinetics and hexanoate oxidation, neonatal piglets were fitted with infusion and sampling catheters (see below). On the morning of surgery (~0900), six normal-weight commercial crossbred piglets from a litter born the previous night were randomly selected, transported to the lab, and maintained in boxes with access to a heat lamp. Surgeries were completed by 1800 when animals were ~15-21 h old, and piglets were fasted until initiation of experiments the following afternoon at 1500 (~36-42 h of age; mass 1.55 ± 0.09 kg). Animals were provided with 20-30 ml of water by gastric intubation the evening before and morning of infusions.For surgery, piglets were anesthetized with 2% isoflurane (Anaquest, Madison, WI) in O2 (15). A 31/2 French umbilical catheter (Sherwood Medical, St. Louis, MO) was inserted into the umbilical artery and advanced 22 cm up the abdominal aorta so that the tip was located at the aortic arch (23). The catheters were subsequently flushed with heparinized physiological saline (5 U/ml) and secured to the umbilicus using suture. This line was used for infusion. A blood sampling catheter was inserted into the left jugular vein to the level of the heart via a cut down to this vessel (38). All procedures were carried out under semisterile conditions using pividone-iodine 10% and ethanol sterilization of surgical fields and instruments. Both catheters were placed along the back of the animal and secured using surgical wrap and tape. Catheters were maintained with heparinized saline, and internal placement of each catheter was confirmed by visual inspection upon termination of the experiment.
Infusion protocol. The aim of this
study was to determine steady-state, nonfermentative acetate
Ra, as well as alterations of
acetate Ra induced by
mitochondrial MCFA -oxidation in neonatal fasted piglets.
Piglets from a given litter were randomly assigned to receive a
continuous infusion of a tracer dose of sodium
[3H]acetate (controls;
ICN Biomedicals, Irvine, CA) or sodium
[3H]acetate coinfused
with sodium
[1-14C]hexanoate (3 piglets/treatment). The experiment was replicated using two litters on
separate days for a total of six piglets per treatment. Animals were
placed in a warmed six-chamber metabolic apparatus, and catheters were
connected to lines exiting the chambers through air-tight septa (14).
The infusion (umbilical) lines were attached to an eight-channel
peristaltic pump (Cole-Parmer, Niles, IL) set to deliver 10 ml/h from
attached intravenous bags (Baxter Healthcare, Deerfield, IL) containing
infusate. Animals were infused with physiological saline for a 1-h
equilibration period before initiation of tracer continuous infusion.
Blood was sampled at intervals from the jugular catheter (limited to 13 ml withdrawn/pig for the entire experiment). Blood was maintained on
ice and promptly centrifuged at 4°C to obtain plasma, which was
frozen at
75°C.
Preliminary experiments indicated that plasma acetate specific activity
(SA) in fasted piglets reaches steady state by 120 min upon initiation
of a continuous infusion of sodium
[3H]acetate (not
shown), so blood collection for determination of acetate SA (see below)
was begun at 90 min of infusion. Collections were then carried out at
130, 170, 210, 250, 290, 330, 370, and 410 min, which corresponded to
the midpoint of each 20-min CO2 collection (see below). Animals were infused for 420 min with a tracer
(48 µCi · h1 · kg
1)
of sodium [3H]acetate
in physiological saline or coinfused with sodium
[3H]acetate plus
sodium
[1-14C]hexanoate
(American Radiolabeled Chemicals, St. Louis, MO) tracer diluted with
nonradioactive sodium hexanoate (SA of the latter was 226 µCi/mol,
delivered at 0.74 µCi · h
1 · kg
1).
Sodium hexanoate substrate was prepared by neutralizing the free acid
of hexanoate (Sigma, St. Louis, MO) with an equimolar amount of NaOH in
water at a final concentration of 474 mM and adjusted to pH 7.4. The
infusion rate of sodium hexanoate (3.27 mmol · h
1 · kg
1)
was estimated to provide 100% of the expected metabolic rate (MR; 141 mmol
ATP · h
1 · kg
1 = 11.2 kJ · h
1 · kg
1;
see Refs. 14 and 23) of a typical neonatal MCFA-infused piglet weighing
1.5 kg, if all hexanoate were completely catabolized to
CO2. Oxidation and infusion rates
of hexanoate in energy equivalents were calculated assuming a
stoichiometry of 44 mmol ATP/mmol hexanoate and 79.5 J/mmol ATP (23).
The 7-h time frame was chosen because steady-state oxidation of MCFA in
fasted piglets is typically within 5 h of continuous infusion of MCFA
(23).
Acetate SA and turnover. SA of plasma
acetate was determined in two steps. Plasma acetate concentrations were
determined by the GLC protocol described above. Samples were
subsequently maintained at 4°C in crimp-top sealed vials until HPLC
separation of acetate was performed 24 h later. Separation of
radiolabeled acetate from other plasma metabolites (i.e., ketone
bodies) was achieved through reversed-phase HPLC using chromatography
conditions identical to those used previously (1, 20). Samples
(40-45 µl) were manually loaded onto a 50-µl injection loop of
the HPLC (Beckman System Gold HPLC Workstation; Beckman Instruments,
San Ramon, CA) and subjected to chromatographic separation using
isocratic elution with 0.3%
H3PO4,
pH 2.1 (flow rate 0.65 ml/min), and a Beckman Ultrasphere IP column (5 µm, 4.6 × 250 mm). For each sample, the eluent fraction
containing acetate (1, 20) was collected into a scintillation vial
using a fraction collector (ISCO Retriever II, Lincoln, NE), and
3H radioactivity was determined
after mixture with 5 ml scintillation cocktail (Bio-Safe II; Research
Products International). Two additional fractions of equal volume were
obtained at the beginning (2-3.5 min) and end (33-34.5 min)
of each 40-min run and served as blanks. Radioactivity was measured
concurrently in the 3H and
14C windows of a Beckman LS 6000IC
scintillation spectrometer. In data not shown, it was demonstrated that
determinations of 3H radioactivity
in acetate were not affected by
[14C]hexanoate
radioactivity because 1) in a subset
of samples derived from hexanoate-infused animals, one distinct
radioactive peak (visualized with in-line scintillation spectrometry;
Radiomatics Flo-one; Packard Instruments, Meridian, CT) was observed
with a retention time corresponding to acetate, and
2)
14C radioactivity values recorded
in the acetate eluent fraction from samples derived from
hexanoate-infused animals were negligible, as determined using
dual-isotope counting procedures.
Steady-state Ra of acetate
(µmol · min1 · kg
1)
was calculated using an equation based on single-pool metabolite
kinetics and using the constant infusion technique (39), calculated as
![]() |
(1) |
CO2 collections and hexanoate oxidation
rate.
The rate of hexanoate oxidation to
CO2 was assessed by measuring the
quantity and SA of expired CO2,
assuming the 1-14C-labeled
carboxyl carbon marked the fate of the entire fatty acid molecule. As
detailed elsewhere (14), air (2 l/min) was drawn through each
respiration chamber after being cleared of atmospheric
CO2 via a sodasorb canister. At
selected intervals, expired CO2
was collected over 20 min and trapped in 75 ml of 1.8 M NaOH. Between
CO2 collections, airflow was
diverted to empty columns, and NaOH was retrieved and replaced in the
trapping columns. Collections were made during the following intervals
after commencement of continuous tracer infusion (in minutes):
80-100, 120-140, 160-180, 200-220, 240-260,
280-300, 320-340, 360-380, and 400-420. The total
amount of trapped CO2 over 20 min
and its SA (µCi/µmol) were determined after
BaCO3 precipitation of NaOH
aliquots, and CO2 production rate
(CO2, µmol/min) was
calculated. The CO2 content of air
collected over 20 min from an empty metabolic chamber served as a
blank. A transfer quotient (TQ), defined as the fraction of
CO2 derived from the oxidation of
hexanoate, was estimated from SA values as follows
![]() |
(2) |
![]() |
(3) |
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Metabolites
Ketone bodies and acetate. Plasma acetate concentrations in piglets (Table 1) were between one and two orders of magnitude higher than total ketone body concentrations over the first 48 h of life, with ketone bodies remaining remarkably low (<40 µM) regardless of suckling status. There was a significant rise in ketone bodies with suckling and age in piglets. Mean circulating acetate concentrations in suckled rat neonates aged 24 h (77 ± 9 µM) and 48 h (175 ± 41 µM) were lower (P < 0.05) than those determined for age-matched piglets (Table 1) and were of similar magnitude to those reported previously for rat pups (4, 16). Acetate concentration in piglets was not significantly different across sampling times.
|
Acetate Kinetics and Hexanoate Oxidation
Acetate Ra and potential contribution to
MR.
The temporal changes in plasma acetate SA in control and
hexanoate-infused piglets are illustrated in Fig.
1A.
Steady-state acetate turnover was achieved by 130 min, with
individual coefficients of variation for steady-state SA averaging 8.9 ± 0.7%. It is notable that one hexanoate-infused piglet
(animal 4-2) displayed a
steady-state acetate SA markedly lower (~3-fold) than its
counterparts (not shown), reflective of a much higher acetate
Ra for that individual (Table
2). Despite this fact, mean acetate
Ra in piglets oxidizing hexanoate
remained just one-half that determined in controls (Table 2; mean
Ra for hexanoate-infused piglets
drops to 40% of controls if animal
4-2 is excluded). Plasma acetate concentrations
during the period of steady state for acetate were equivalent
(P > 0.1) in controls (79 ± 4 µM) and hexanoate-infused piglets (109 ± 23 µM).
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
An increasing body of evidence supports the idea that certain aspects of fatty acid metabolism in the neonatal piglet stray significantly from traditional paradigms. Of particular note is the ketogenic deficit in piglets (1, 2, 8, 20, 24, 28), which would be expected to promote "alternative," nonketogenic pathways of fuel carbon flow in the liver. Our efforts to characterize such phenomena through analyses of metabolic end products of hepatic fatty acid oxidation in vitro have demonstrated that ketogenesis is supplanted by acetogenesis and other routes that await further clarification (1, 20, 24). The current set of experiments represents a first attempt to pinpoint the relevance of acetogenesis vs. ketogenesis for neonatal piglets in vivo and to probe the analogy between ketogenesis and acetogenesis with enhanced provision of fatty acids in these animals.
Acetate Metabolism During the Neonatal Period
The data presented herein are fully consistent with an important physiological role for acetate (e.g., as a fuel source or lipid precursor) in neonatal piglets, especially in light of the minimal metabolic impact of ketone bodies in this model (38). Circulating acetate concentrations over the first 2 days of life, for instance, are one to two orders of magnitude higher than ketone bodies in piglets (Table 1). In contrast, postnatal acetate values in ketogenic rat pups (~100-200 µM; also see Refs. 4 and 16) are ~10-fold lower than the ketone body levels reported for these neonates (see Ref. 10). Whether comparisons of circulating acetate and ketone body levels reflect differences in utilization of these metabolites in piglets could not be directly assessed from plasma concentration alone. However, we have previously determined that, in the physiological range, plasmaAlthough the kinetics of ketone bodies in piglets have not been
described, the substantial Ra of
endogenously produced acetate in fasting piglets (22-51
µmol · min1 · kg
1;
Table 2) rivals that of glucose in 48-h-old fasted piglets (23-31
µmol · min
1 · kg
1;
see Refs. 29 and 30). This signals the presence of an active acetogenic
pathway in vivo, which might support interorgan fuel carbon
trafficking. Should steady-state
Ra values for acetate (Table 2)
and glucose (29, 30) reflect oxidative disposal in fasted piglets, then
acetate potentially met ~20% (Table 2), and glucose 45-60%, of
the ATP requirements of piglets in the current study. The significant
energy potential of fuels such as glucose, amino acids, and perhaps
acetate may be especially important to the survival of fasting neonatal
piglets, in which circulating NEFA (27, 37) and ketone bodies (Table 1;
Refs. 2 and 27) are trifling.
Finally, it is relevant to note that energy potential calculations for acetate (Table 2) would be shifted downward if cycling between acetate and acetyl-CoA (6) is significant in piglets. Cycling mandates that multiple rounds of acetate activation occur before combustion; thus, net ATP yield per mole of acetate oxidized would fall concomitant with the degree of cycling. In control piglets, estimates of acetate oxidation as a percentage of MR (Table 2) would also be affected by differences in RQ assumptions, becoming smaller with a lower RQ. Research defining true flux of acetate carbon to CO2 in vivo (and studies examining the magnitude of acetate/acetyl-CoA cycling) will be required to fully define the contribution of acetate oxidation toward meeting the fasting or suckling piglet's energy demands.
Etiology of Endogenous Acetogenesis in Piglets
Biogenesis of acetate, occurring in a variety of mammalian tissues, is thought to be contingent upon relative activities of acyl-CoA hydrolases and synthetases toward acetyl-CoA and acetate, respectively (4, 6, 7, 16). Although control over acetogenesis is poorly understood, acetyl-CoA hydrolysis might be expected to rise concomitant with an increase in substrate availability. Descriptions of fatty acid metabolism typically emphasize increased acetyl-CoA availability concurrent with elevated fatty acidMCFA bypass regulation of -oxidation at the level of mitochondrial
CPT-I by virtue of their diffusibility, thus eliciting a robust
ketogenic response in a number of animals in vivo, but not in piglets
(2). Indeed, when piglets were infused with MCFA at maximal rates (23),
steady-state ketone body concentrations never exceeded 0.1 mM and could
not account for >2-3% of energy expenditure (38). We therefore
hypothesized that, in the piglet, abundant provision of hexanoate would
stimulate mitochondrial acetogenesis if tissue acetate production
emulates hepatic ketogenesis. On the contrary, despite active
combustion of hexanoate (Fig. 1 and Table 3), acetate
Ra was curtailed to a level just
one-half that of controls (Table 2). This result clearly indicates that improved fatty acid substrate availability is not associated with accelerated acetate production at this rate of hexanoate delivery in
piglets, and thus under these conditions acetogenesis does not mimic
ketogenesis.
Although any explanation for these results is tentative, some interesting observations about piglet metabolism warrant consideration. Exposure of piglet hepatocytes to 1 mM MCFA in vitro elicits a drop of up to 50% in total hepatocyte acetyl-CoA concentration (20). Furthermore, in vivo infusion of octanoate to piglets at a rate similar to that of hexanoate in the current work resulted in a diminution of hepatic acetyl-CoA levels to 15% of control values (15). It is thus plausible that a drop in hepatic or extrahepatic tissue acetyl-CoA concentration in animals infused with hexanoate could have occurred, thereby limiting acetyl-CoA hydrolase substrate availability and/or altering the dynamics of acetate/acetyl-CoA cycling. Further studies will be required to assess how changes in fatty acid availability impact tissue acetyl-CoA levels in pigs and to ascertain if such changes affect acetogenesis.
The fact that oxidation of hexanoate resulted in marked changes in
acetogenesis (Table 2) adds to a growing body of evidence supporting an
important role for mitochondria in acetate generation in the piglet.
Acetyl-CoA hydrolase activity has been demonstrated in liver, heart,
and kidney mitochondria of pigs (18, 22; X. Lin and J. Odle,
unpublished results). In addition, chain-length restrictions for
peroxisomal -oxidation (17, 25) dictate that hexanoate is an
exclusively mitochondrial fuel, yet this and other MCFA clearly impact
acetogenesis in piglets (this study and also Ref. 20) and rat tissues
(19, 34, 35). Finally, inclusion of tricarboxylic acid cycle or
-oxidation inhibitors with incubations of piglet liver preparations
modulates acetogenesis from fatty acids upward and downward,
respectively (1, 20). The cytosol (7) and peroxisomes (13, 19, 25) of
tissues also carry out acetogenesis, but their contribution to acetate
production in the current study is not known.
In conclusion, results from studies in piglets suggest that acetogenesis must be considered when attempting to optimize the current understanding of neonatal metabolism. The basis for the metabolic profile of piglets remains to be fully elucidated, but unique characteristics of enzyme systems and hormones appear to be involved. For instance, poor ketogenesis in piglets is likely related to the lack of a postnatal induction of mitochondrial HMG-CoA synthase in which hepatic activity (3, 8) and mRNA abundance in liver (and small intestine; see Ref. 3) remain negligible, contrasting with observations in neonatal rats (10, 11, 32). The postnatal induction of HMG-CoA synthase and liver CPT-I gene expression/activity in rats is supported by the precipitous and stable drop in the insulin-to-glucagon ratio after birth (10, 32). In the piglet, initiation of suckling over the first days of life markedly increases plasma insulin (27, 37), which may influence fuel carbon flux through alterations in gene expression and/or more direct effects upon enzyme activities.
The prospect that acetate more profoundly impacts piglet metabolism vs.
ketone bodies is supported by the results of this study. Like ketone
bodies, acetate could represent a viable fatty acid precursor
and/or fuel source for developing piglets. With respect to its
production, on the other hand, the analogy between nonfermentative
acetogenesis and ketogenesis is not strong under the conditions tested.
For example, initiation of suckling in rats is associated with a sharp
rise in blood ketone bodies concomitant with increased fatty acid
availability and -oxidation (10). Acetate levels in piglet plasma
were equivalent regardless of age or nutritional status (Table 1), and
acetate Ra was substantial in
animals fasted
30 h (Table 2), despite large differences in plasma
NEFA concentration reported for newborn, suckled, and fasted piglets
(27, 37). Numerous acetyl-CoA generators, notably pyruvate and lactate,
could give rise to acetate. These metabolites are relatively abundant
in the blood of suckling (pyruvate, ~250 µM; lactate, ~3 mM) or
fasting (pyruvate, ~500 µM; lactate, ~5 mM) piglets (27),
possibly maintaining acetate production independent of tissue fatty
acid
-oxidation. Indeed, a dissociation between acetogenesis and
ketogenesis has been reported when ketogenic flux from LCFA was altered
over a wide range in perfused rat liver (9, 36). Thus it appears that
endogenous mitochondrial acetogenesis may represent a pathway in which
operation is dictated largely by maintenance of an adequate acetyl-CoA
pool, independent of its source.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Amy Reeder, Dr. Xi Lin, and Dr. Ruurd Zijlstra for technical assistance. Dr. Sharon Donovan (Div. of Nutritional Sciences, University of Illinois at Urbana-Champaign) kindly provided neonatal rat serum samples.
![]() |
FOOTNOTES |
---|
This work was supported by United States Department of Agriculture Grant 92-37203-7993 (project 35-0525).
Current address of S. H. Adams: Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080.
Address for reprint requests: J. Odle, Dept. of Animal Sciences, North Carolina State University, Raleigh, NC 27695-7621.
Received 2 September 1997; accepted in final form 16 February 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Adams, S. H.,
X. Lin,
X. X. Yu,
J. Odle,
and
J. K. Drackley.
Hepatic fatty acid metabolism in pigs and rats: major differences in endproducts, O2 uptake and -oxidation.
Am. J. Physiol.
272 (Regulatory Integrative Comp. Physiol. 41):
R1641-R1646,
1997
2.
Adams, S. H.,
and
J. Odle.
Plasma -hydroxybutyrate after octanoate challenge: attenuated ketogenic capacity in neonatal swine.
Am. J. Physiol.
265 (Regulatory Integrative Comp. Physiol. 34):
R761-R765,
1993
3.
Adams, S. H.,
C. Sampaio Ahlo,
F. G. Hegardt,
and
P. F. Marrero.
Gene expression of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in a poorly ketogenic mammal: effect of starvation during the neonatal period of the piglet.
Biochem. J.
324:
65-73,
1997[Medline].
4.
Buckley, B. M.,
and
D. H. Williamson.
Origins of blood acetate in the rat.
Biochem. J.
166:
539-545,
1977[Medline].
5.
Chatelain, F.,
C. Kohl,
V. Esser,
J. D. McGarry,
J. Girard,
and
J.-P. Pégorier.
Cyclic AMP and fatty acids increase carnitine palmitoyltransferase I gene transcription in cultured fetal rat hepatocytes.
Eur. J. Biochem.
235:
789-798,
1996[Abstract].
6.
Crabtree, B.,
S. A. Marr,
S. E. Anderson,
and
J. C. MacRae.
Measurement of the rate of substrate cycling between acetate and acetyl-CoA in sheep muscle in vivo.
Biochem. J.
243:
821-827,
1987[Medline].
7.
Crabtree, B.,
M. Souter,
and
S. E. Anderson.
Evidence that the production of acetate in rat hepatocytes is a predominantly cytoplasmic process.
Biochem. J.
257:
673-678,
1989[Medline].
8.
Duée, P.-H.,
J.-P. Pégorier,
P. A. Quant,
C. Herbin,
C. Kohl,
and
J. Girard.
Hepatic ketogenesis in newborn pigs is limited by low mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase activity.
Biochem. J.
298:
207-212,
1994[Medline].
9.
Frölich, J.,
and
O. Wieland.
Dissoziierung von ketogenese und acetatbildung in der isoliert perfundierten rattenleber (Abstract).
Z. Klin. Chem. Biochem. Suppl.
6:
241,
1975.
10.
Girard, J.,
P. Ferré,
J.-P. Pégorier,
and
P.-H. Duée.
Adaptations of glucose and fatty acid metabolism during perinatal period and suckling-weaning transition.
Physiol. Rev.
72:
507-562,
1992
11.
Hegardt, F. G.
Regulation of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene expression in liver and intestine from the rat.
Biochem. Soc. Trans.
23:
486-490,
1995[Medline].
12.
Herpin, P.,
J. LeDividich,
D. Berthon,
and
J.-C. Hulin.
Assessment of thermoregulatory and postprandial thermogenesis over the first 24 hours after birth in pigs.
Exp. Physiol.
79:
1011-1019,
1994[Abstract].
13.
Hovik, R.,
B. Brodal,
K. Bartlett,
and
H. Osmundsen.
Metabolism of acetyl-CoA by isolated peroxisomal fractions: formation of free acetate and acetoacetyl-CoA.
J. Lipid Res.
32:
993-999,
1991[Abstract].
14.
Kempen, T. A. T. G.,
and
J. Odle.
Medium-chain fatty acid oxidation in colostrum-deprived newborn piglets: stimulative effect of L-carnitine supplementation.
J. Nutr.
123:
1531-1537,
1993[Medline].
15.
Kempen, T. A. T. G.,
and
J. Odle.
Carnitine affects octanoate oxidation to carbon dioxide and dicarboxylic acids in colostrum-deprived piglets: in vivo analysis of mechanisms involved based on CoA- and carnitine-ester profiles.
J. Nutr.
125:
238-250,
1995[Medline].
16.
Knowles, S. E.,
I. G. Jarret,
O. E. Filsell,
and
F. J. Ballard.
Production and utilization of acetate in mammals.
Biochem. J.
142:
401-411,
1974[Medline].
17.
Lazarow, P. B.
Rat liver peroxisomes catalyze the -oxidation of fatty acids.
J. Biol. Chem.
253:
1522-1528,
1978[Abstract].
18.
Lee, K. Y.,
and
H. Schulz.
Isolation, properties, and regulation of a mitochondrial acyl coenzyme A thioesterase from pig heart.
J. Biol. Chem.
254:
4516-4523,
1979[Medline].
19.
Leighton, F.,
S. Bergseth,
T. Rørtveit,
E. N. Christiansen,
and
J. Bremer.
Free acetate production by rat hepatocytes during peroxisomal fatty acid and dicarboxylic acid oxidation.
J. Biol. Chem.
264:
10347-10350,
1989
20.
Lin, X.,
S. H. Adams,
and
J. Odle.
Acetate represents a major product of heptanoate and octanoate -oxidation in hepatocytes isolated from neonatal piglets.
Biochem. J.
318:
235-240,
1996[Medline].
21.
Lopes-Cardozo, M.
Regulation of ketogenesis in isolated rat liver mitochondria.
In: Biochemical and Clinical Aspects of Ketone Body Metabolism, edited by H. D. Söling,
and C. D. Seufert. Stuttgart: Georg Thieme Publishers, 1978, p. 23-40.
22.
Markwell, M. K.,
E. J. McGroarty,
L. L. Bieber,
and
N. E. Tolbert.
The subcellular distribution of carnitine acyltransferases in mammalian liver and kidney.
J. Biol. Chem.
248:
3426-3432,
1973
23.
Odle, J.,
N. J. Benevenga,
and
T. D. Crenshaw.
Evaluation of [1-14C]-medium-chain fatty acid oxidation by neonatal piglets using continuous-infusion radiotracer kinetic methodology.
J. Nutr.
122:
2183-2189,
1992[Medline].
24.
Odle, J.,
X. Lin,
T. A. T. G. van Kempen,
J. K. Drackley,
and
S. H. Adams.
Carnitine palmitoyltransferase modulation of hepatic fatty acid metabolism and radio-HPLC evidence for low ketogenesis in neonatal pigs.
J. Nutr.
125:
2541-2549,
1995[Medline].
25.
Osmundsen, H.,
C. E. Neat,
and
B. Borrebaek.
Fatty acid products of peroxisomal -oxidation.
Int. J. Biochem.
12:
625-630,
1980[Medline].
26.
Palmquist, D. L.
Palmitic acid as a source of endogenous acetate and -hydroxybutyrate in fed and fasted ruminants.
J. Nutr.
102:
1401-1406,
1972[Medline].
27.
Pégorier, J.-P.,
P.-H. Duée,
R. Assan,
J. Peret,
and
J. Girard.
Changes in circulating fuels, pancreatic hormones and liver glycogen concentration in fasting or suckling newborn pigs.
J. Dev. Physiol. (Eynsham)
3:
203-217,
1981[Medline].
28.
Pégorier, J.-P.,
P.-H. Duée,
J. Girard,
and
J. Peret.
Metabolic fate of non-esterified fatty acids in isolated hepatocytes from newborn and young pigs.
Biochem. J.
212:
93-97,
1983[Medline].
29.
Pégorier, J.-P.,
P.-H. Duée,
C. Simoes-Nunes,
J. Peret,
and
J. Girard.
Glucose turnover and recycling in unrestrained and unanesthetized 48-h-old fasting or postabsorptive newborn pigs.
Br. J. Nutr.
52:
277-287,
1984[Medline].
30.
Pégorier, J.-P.,
C. Simoes-Nunes,
P.-H. Duée,
J. Peret,
and
J. Girard.
Effect of intragastric triglyceride administration on glucose homeostasis in newborn pigs.
Am. J. Physiol.
249 (Endocrinol. Metab. 12):
E268-E275,
1985
31.
Pouteau, E.,
H. Piloquet,
P. Maugeais,
M. Champ,
H. Dumon,
P. Nguyen,
and
M. Krempf.
Kinetic aspects of acetate metabolism in healthy humans using [1-13C]acetate.
Am. J. Physiol.
271 (Endocrinol. Metab. 34):
E58-E64,
1996
32.
Prip-Buus, C.,
S. Thumelin,
F. Chatelain,
J.-P. Pégorier,
and
J. Girard.
Hormonal and nutritional control of liver fatty acid oxidation and ketogenesis during development.
Biochem. Soc. Trans.
23:
500-506,
1995[Medline].
33.
Rémésy, C.,
and
C. Demigné.
Determination of volatile fatty acids after ethanolic extraction.
Biochem. J.
141:
85-91,
1974[Medline].
34.
Seufert, C. D.,
M. Graf,
G. Janson,
A. Kuhn,
and
H. D. Söling.
Formation of free acetate by isolated perfused livers from normal, starved and diabetic rats.
Biochem. Biophys. Res. Commun.
57:
901-909,
1974[Medline].
35.
Seufert, C. D.,
K. P. Grigat,
K. Koppe,
and
H. D. Söling.
Regulation of formation of ketone bodies and acetate in rat liver.
In: Biochemical and Clinical Aspects of Ketone Body Metabolism, edited by H. D. Söling,
and C. D. Seufert. Stuttgart: Georg Thieme Publishers, 1978, p. 23-40.
36.
Snoswell, A. M.,
R. P. Trimble,
R. C. Fishlock,
G. B. Storer,
and
D. L. Topping.
Metabolic effects of acetate in perfused rat liver. Studies on ketogenesis, glucose output, lactate uptake and lipogenesis.
Biochim. Biophys. Acta
716:
290-297,
1982[Medline].
37.
Swiatek, K. R.,
D. M. Kipnis,
G. Mason,
K. Chao,
and
M. Cornblath.
Starvation hypoglycemia in newborn pigs.
Am. J. Physiol.
214:
400-405,
1968[Medline].
38.
Tetrick, M. A.,
S. H. Adams,
J. Odle,
and
N. J. Benevenga.
Contribution of D-()-3-hydroxybutyrate to the energy expenditure of neonatal pigs.
J. Nutr.
125:
264-272,
1995[Medline].
39.
Wolfe, R. R.
Radioactive and Stable Isotope Tracers in Biomedicine. New York: Wiley-Liss, 1992.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |