T H EJOURNAL OF BIOLOGICAL CHEMISTRY Vol . 266, No. Issue of April 15, pp.

6985-6990,1991
1991 by The American Society for Biochemistry and Molecular Biology, Inc
IC.
Printed in U.S.A.

Metabolism of [2-14C]Acetate andIts Use in Assessing Hepatic Krebs

Cycle Activity and Gluconeogenesis*
(Received for publication, July 3, 1990)

William C. Schumannf, Inger Magnussons,Visvanathan Chandramoulif, Kozhikot Kumaranf,

John Wahrenj, and Bernard R. Landaufll
From the $Departments of Medicine and Biochemistry, Case Western Reserve Uniuersity, Cleveland, Ohio 44106 and the
§Departments of Clinical Physiology, Karolinska Institute, Huddinge Hospital, s-141 86 Huddinge and Karolinska Hospital,
S-104 01 Stockholm, Sweden

To examine the fate of the carbons of acetate and to acetate in the carbons of glucose from blood and of glutamate
evaluate the usefulness of labeled acetate in assessing from urinary phenylacetylglutamine under the same condi-
intrahepatic metabolic processes during gluconeogen- tions as for [3-I4C]lactate. Thisallows a direct comparison of
esis, [2-14C]acetate,[2-14C]ethanol,and [l-14C]ethanol the fate of the carbons of acetate and lactate and tests the
were infused into normal subjects fasted 60 h and given validity of using [2-'4C]acetate, as hasbeen done (8-11, 13).
phenyl acetate. Distributions of 14C in the carbons of
blood glucose and glutamate from urinary phenylace- EXPERIMENTALPROCEDURES
tylglutamine were determined. With [2-'4C]acetate
and [2-'4C]ethanol,carbon 1 of glucose had abouttwice Subjects-Six healthy women (ages 25-41 y and weighing 54-75
as much I4C as carbon 3. Carbon 2 of glutamate had kg) were studied. They consumed a diet containing at least 200 g of
about twice as much 14C as carbon 1 and one-half to carbohydrate for a t least 3 days before fasting. None were taking any
medication. The experimental protocol was approved by the Human
one-third as much as carbon 4. There was only a small Investigation Committees a t Huddinge University Hospital and Uni-
amount in carbon 5. These distributions are incompat- versity Hospitals of Cleveland. Informed consent was obtained from
ible with the metabolism of [2-14C]acetatebeing pri- each subject.
marily in liver. Therefore, [2-14C]acetate cannot be Procedure-Thesubjects were studiedafter 60 h of fastingas
used to study Krebs cycle metabolism in liver and in previously described for [3-"C]lactate administrations (1).Each sub-
relationship to gluconeogenesis, as has been done. The ject ingested1.6 or 1.9 g of sodium phenyl acetate a t 10-15-min
distributions can be explained by: (a)fixation of l4CO, intervals for a total of 4.8 or 5.7 g (30 or 36 mmol). Each portionwas
from [2-14C]acetatein the formation of the 14C-labeled dissolved in Coca-Cola Light' (Pripps Brewery, Bromma, Sweden) to
glucose and glutamate in liver and ( b )the formation of mask its taste. At the time thesecond portion of phenyl acetate was
ingested, an infusion of a 14CC-labeled compound through a peripheral
14C-labeledglutamate in a second site, proposed to be vein was begun a t a constant rate and was continued for 6 h. Four
muscle. [ 1,3-'4C]Acetone formation from the [2-14C] subjects were given [2-"C]acetate, one [2-"C]ethanol, and one [ I -
acetate does not contribute to the distributions, as evi- "C]ethanol. Sixty mlof blood was drawn from another peripheral
denced by the absence of 14C in carbons 2-4 of gluta- vein every 1.5 h, and urine was collected in 1.5-h intervals throughout
mate after [ l-'4C]ethanol administration. the period of infusion.
The "C-labeled substrates were given in trace amounts;40 pCi was
infused into each subject. Each subject, throughout the 6-h of infu-
sion, was encouraged to drink 240 ml of water every hour.
Materials-Sodium phenyl acetate was prepared as previously de-
A model of Krebs cycle metabolism and gluconeogenesis scribed (1).Sodium [2-"C]acetate, [2-14C]ethanol, and [l-"C]ethanol
has been described and tested (1). Relative flow rates and were purchased fromAmersham International(Buckinghamshire,
carbon exchange in the Krebs cycle were calculated from the England). [P-"C]Acetate gave a single peak of "C with the mobility
distribution of "C in the carbon of glutamate from urinary of acetate on HPLC' using an Aminex HPX-87H column (Bio-Rad)
phenylacetylglutamine after administering 13-14C]lactate and a t room temperature with 0.01 N H,SO, as solvent. [l-"'C]Ethanol
phenylacetate.Comparingthedistributions of in blood and[2-''C]ethanol were, beyond the evidence of purity from the
manufacturer, subjected to HPLC using a fermentation monitoring
glucose and the glutamate and their specific activities pro- column (Bio-Rad) with 0.002 N HLSOJ as solvent a t 65 "C. [l-"C]
vided evidence for thedistributioninglutamate reflecting Ethanol had a 5%14C-labeledimpurity that co-migrated with acetate
that in hepatic a-ketoglutarate. on HPLC, and [2-'4C]ethanol had a 3% labeled impurity that was
[2-"C]Acetate has been proposed as a substrate for deter- not further defined.
mining the correction factor for carbon exchange in the Krebs Analyses-Plasma glucose concentration was determined enzymat-
cycle in liver during gluconeogenesis (2-7) and has beenused ically (14). From the remainder of the blood samples, glucose was
isolated and degraded (15, 16) to obtain the percent distribution of
in studies in vivo in rats (8) and dogs (9) and humans (10- IJC in each of its carbons. Eachof the glucoses degraded had between
13). The purpose of this study was to investigate the fate of 2000 and 5000 dpm of "C.
acetate by determining the distribution ofI4C from [2-"C]- Phenylacetylglutamine, e-hydroxybutyrate, andurea were isolated
from urine (1). Thespecific activities of e-hydroxybutyrate and urea
* This work was supported by National Institutes of Health Grant were determined (1). Glutamic acid from phenylacetylglutamine was
DK-14.507 and Grant3108 from the Swedish Medical Research Coun- isolated and degraded to obtain the percent distribution of "C in each
cil. The costs of publication of this article were defrayed in part by of its carbons (1). Between 1.8 and 3.2 mmol of glutamic acid was
the payment of page charges. This article must therefore be hereby isolated from each sample, and between 9000 and 40,000 dpm was
marked "advertisement" in accordance with 18 U.S.C. Section 1734 used in each degradation.
solely to indicate this fact. Calculations-To correct the distributionsin glutamate andglucose
ll Recipient of a John F. Fogarty Senior International fellowship.
To whom correspondenceshould be addressed: Dept. o f Medicine, ' The abbreviationused is: HPLC, high pressure liquid chromatog-
University Hospitals, 2074 Abington Rd., Cleveland, OH 44106. raphy.

6985
6986 Metabolism of Acetate
for incorporation via fixation of I4CO2formed from the [2-I4C]acetate, "Experimental Procedures" and the data from Table I11 as
recourse was made to the method previously reported (1). The expres- shown in Ref. 1. Corrections were 38, 29, and 21% in carbon
sions used were:
1of glutamate in the 1.5-3-, 3-4.5-, and 4.5-6-h urine samples
['%]ureaA and 60 and 61% in carbons3 and 4 of glucose at 4.5 and 6 h,
['4C]GlutamateA= X [I4C]glutamateB (1)
[I4C]ureaB respectively.
Plasma glucose concentration (mean f S.E.), after 60 h of
[14C]ureaA
[14C]G1~coseA
= X [14C]glucoseH fasting, was 3.8 f 0.4 mmol/liter and, after 6 h of infusion,
[I4C]ureaB 3.2 f 0.2 mmol/liter.
where ["C]glutamateA and [14C]glutamateBare the 14C specific ac-
tivities of glutamate due to 14C02fixation on administering [2-I4C] DISCUSSION
acetate and ['%]bicarbonate, respectively; [14C]gl~coseA and ["C]
Based on the distribution of14C inglutamate from the
glucoseHare the corresponding specific activities of glucose; and ["C]
glutamineconjugate of phenylacetate,[3-'4C]lactatehas
ureaAand ["C]urea~are the corresponding specific activities of urea.
proven to bea satisfactory substrate for estimating the rates
RESULTS of the different reactions involved in the Krebs cycle and in
gluconeogenesis (Table VI11of Ref. 1). Theseestimations
The percent incorporations of 14C into the carbons of glu-
were possible because apparently themajor portion of the [3-
tamate (designated by K with subscripts) from urinary phen-
14C]lactate yielding those distributions was metabolized di-
ylacetylglutamine excreted by each subject are recorded in
rectly in liver. Therefore, the distribution in the glutamate
Table I. With [2-14C]acetate and [2-14C]ethanol administra-
was representative of events occurring in liver per se. The
tion, carbon 4 had the most 14C(41.0-52.3%). Carbons 2 and
situation is very different with [2-I4C]acetate.
3 had similar amounts (15.8-24.0%), and carbon 1 had about
half the "C activity of carbons 2 and 3 (9.4-15.8%). Carbon We propose, as illustrated in Fig. 1, that in contrast to the
5 had 0.2-5.9%. [l-'4C]Ethanol administration resulted in I4C metabolism of [3-'4C]lactate, there isconsiderable oxidation
incorporation in carbons 1 and 5 , with -3 times as much in of [2-I4C]acetate to 14C02 in muscle and that a substantial
carbon 5 as in carbon1. portion of labeled glutamate is formed from [2-14C]acetatein
Distributions of 14C in the carbons of blood glucose (desig- muscle, converted to glutamine,conjugated with phenyl ace-
nated by G with subscripts) from the subjects given [2-"C] tate, and excreted in urine. Thus, the distribution of14C in
acetate and [2-I4C]ethanol areshown in Table 11. Carbons 1, the glutamine is not representative of the eventsoccurring in
2, 5 , and 6 have similar percentages (17.4-22.2%). Carbons 3 liver per se, but a combination of events occurring in periph-
and 4 have somewhat more than half the 14Cincorporations eral tissue and liver. It is for this reason that the14Cpattern
of the other carbons(10.1-12.5%). of the glutamate (a-ketoglutarate) is not a reflection of the
Specific activities of urinary urea, glutamate from urinary I4C pattern of the oxalacetate of liver from which glucose is
phenylacetylglutamine, urinary P-hydroxybutyrate,blood glu- formed via P-enolpyruvate. Furthermore, estimates that have
cose, and expired CO, from the subjects given [2-'4C]acetate been made of carbon exchange in the Krebs cycle in liver in
are recordedin Table 111. Recorded in the last column of vivo using [2-'4C]acetate are notvalid. The reasons for these
Table I11 are data from Ref. 1 for subjects given [14C]bicar- conclusions are discussed below.
bonate in the fasted state. Fate of Carbons of Acetate-An abbreviated scheme of the
Table IV gives the mean14Cdistributions in glutamatefrom Krebs cycle and of gluconeogenesis in liver is shown in Fig. 1
urine from the subjects given [2-14C]acetate and the distri- (for a more complete scheme, see Fig. 1of Ref. 1).The carbons
butions correctedfor theincorporation of14C from 14C02 of oxalacetate are designated by 0, a-ketoglutarate by K,
formed from the [2-14C]acetate.The average 14Cdistributions fumarate by F, P-enolpyruvate by E, pyruvate by P, lactate
in blood glucose from the subjects given [2-14C]acetate and by L, and glucose by G. Certain relationships should prevail
the distributions corrected for 14C02 incorporation aregiven if [2-14C]acetate is metabolized in liver per se ina closed
in Table V. Corrections for "CO, incorporation in glutamate system, i.e., with no carbons other than those of the [2-"C]
and glucose were obtained using Equations 1 and 2 under acetyl-coA and unlabeled CO, entering the cycle. If the spe-

TABLE I
Distribution of "C in thecarbons of glutamate from urinary phenylacetylglutamine
Urine "C-Compound "C in carbons
Subject Recovery KJK,
infused collection KI K2 K:, K4 Ks
h 96 96
U.E. [2-"C]Acetate 1.5-3 17.3 9.7 17.2 50.0 5.9 90.5 1.8
4.5-6 9.4 21.5 24.0 42.9 2.1 94.1 2.3

A.J. [2-"C]Acetate 1.5-3 10.1 16.7 15.8

5.0 52.3 101.7 1.7
4.5-6 10.0 22.8 21.7 44.6 1.0 94.4 2.3

M.T. [2-I4C]Acetate 3-4.5 11.8 19.7 20.8 47.0 0.7 83.0 1.7
4.5-6 13.1 21.1 22.0 43.2 0.5 94.0 1.6

K.H. [Z-"C]Acetate 3-4.5 13.8 18.9 0.2

20.6 46.4 87.5 1.4
4.5-6 15.8 19.8 23.0 41.0 0.4 97.6 1.3

M.L.H. [2-"C]Ethanol 4.5-6 12.5 22.6 20.5

1.6 42.8 85.4 1.8

U.H. [l-14C]Ethan~l 0-3 22.7 0.6" 76.9 98.4

3-6 28.0 0.0" 71.8 98.9
'' Value is sum of K, + K,, + K.,.
 Metabolism of Acetate 6987
TABLEI1
Distribution of "C in thecarbons of blood glucose
Time
'%Compound "C in carbons
Subject Recovery G,/G.,
sampled infused G~ G2 G:, G, G6 Gti
h % %
U.E. 4.5
[2-"C]Acetate 18.7 19.3 10.619.8 11.6 20.0 94.2 1.8
6 18.5 19.5 10.9 11.4 18.5 22.2 100.7 1.7

4.5
[2-I4C]Acetate
A.J. 19.5 18.6 10.2 12.0 18.5 21.2 96.9 1.9
6 18.7 18.7 12.1 11.6 19.6 19.3 92.4 1.5

M.T. 4.5
[2-I4C]Acetate 18.1 17.4 10.4 11.1 20.8 22.2 108.6 1.7
6 18.1 18.3 11.6 12.5 19.1 20.2 95.9 1.6

K.H. 6[2-14C]Acetate 18.6 18.8" 11.1 11.9 20.0 19.8 101.4 1.7

[2-'4C]Ethanol
M.L.H. 4.5 19.4 18.0 10.1 11.5 20.9 20.1 96.8 1.9
6 19.0 19.0 10.5 12.2 18.3 21.0 98.2 1.8
I' Corrected for lossof G , in the degradation procedure.

TABLE 111 TABLEIV

Specific activities of urinary urea, glutamate from urinary Mean distribution of 14C in thecarbons of glutamate from urinary
phenylacetylglutamine, urinary ,"hydroxybutyrate, blood glucose, phenylacetylglutamine from subjects given [2-'4C]acetate and those
and expired CO, from fasted subjects given distributions corrected for incorporationof I4CO2
[2-'4C]acetate and r'C1bicarbonate "C in carbons
Time sampled KdKI
Compound
measured s:iFLd Bicarbonate"
Labeled compound"
Acetate
K1 K, K:,
%
Kq K,

h dpmlgmol 1.5-3 9.9 17.0 16.5 51.2 5.5 1.7

Urea 0-1.5 0.04 f 0.01 (4) 0.59 f 0.19 (4) 1.5-3 corrected 6.1 17.7 17.2 53.3 5.7 2.9
1.5-3 0.49 f 0.13 (3) 3.51 f 1.20 (4)
3-4.5 1.56 f 0.26 (4) 7.34 f 0.88 (4) 3-4.5 12.8 19.3 20.7 46.7 0.5 1.5
4.5-6 2.87 f 0.36 (4) 10.5 f 0.86 (4) 3-4.5 corrected 9.1 20.1 21.6 48.7 0.5 2.2

Glutamate 0-1.5 3 f 1 (3) 4.5-6 12.1 21.31.8 22.7

1.0 42.9
1.5-3 *
9 (3)
3 *
3.2 0.5 (4) 4.5-6 corrected 9.5 21.9 23.4 44.2 2.31.0
3-4.5 *
24 5 (3) 4.5 f 0.6 (4)
4.5-6 *
48 3 (4) 5.1 f 0.6 (4)
TABLE V
@-Hydroxybutyrate 3-4.5 41 (1) Mean distribution of I4C in thecarbons of blood glucose from subjects
4.5-6 43 f(3)
9 giuen [2-"C]acetate and those distributions corrected for
incorporation of "CO,
Glucose
4.5 44 f 4
(4) 29 f 3 (4) "C in carbons
6 54 f 4(4)30 f3(4) Time sampled G 1/G.I
GI G2 G:, G, Gs Ge
co, h 76
4.5 18.8 18.4 10.4 11.6 19.7 21.1 1.8
4.5 corrected 21.9 21.5 4.2 4.7 23.0 24.7 5.2

6 18.5 18.8 11.4 11.9 19.3 20.4 1.6

*
'' All values are given as mean S.E. with the numbers in paren-
theses representing numberof subjects.
*Data are fromRef. 1.
cific activity of carbon 2 of acetyl-coA is set 100
to on repeated
cycling, the relative distribution of 14C in oxalacetate will be
O l = 50, 0, = 100, O:, = 100, and O4 = 50 andina-
ketoglutarate K1 = 50, K, = 100, K3 = 100, K4 = 100, and K,
= 0, thus the ratio K2/K,= 2 and K4/KZ= 1.
When gluconeogenesis occurs (the system is open), pyru-
vate is carboxylated to oxalacetate, which is converted to P-
enolpyruvate, the precursor of glucose. The ratios of incor-
porations of14C from [2-I4C]acetate into carbon2 to carbon
1 of a-ketoglutarate (K,/Kl) and carbon 1 to carbon 3 of
glucose (G1/G:{)are a function of the rate of pyruvate carbox-
ylation relative to Krebscycle flux. These ratios canbe <2 in
only two circumstances, because there is 14C0, fixation by
pyruvate and because of [1-14C]acetyl-CoA formation. The
only way [l-'4C]acetyl-CoA can be formed is if the [2,3-14C]
6 corrected 21.8 22.2 4.5 4.6 22.8 24.1 4.8
oxalacetate is convertedvia P-enolpyruvate to [2,3-'*C]pyru-
vate, which is then decarboxylated to [1,2-'4C]acetyl-CoA. 14C
from [l-14C]acetyl-CoA would then be incorporated via the
cycle into carbon 1 of a-ketoglutarate and into carbon 3 of
glucose. Under all circumstances, KP = Ka and G, = GI. If
there is complete isotopic equilibration of oxalacetate with
fumarate, KZ/K1 will equal G1/G:+ The greater the rate of
pyruvatecarboxylation relative toKrebs cycle flux, the
greater the K2/K1 and K4/KP ratios.
Distributions of 14C from [2-14C]Acetate in Glucose and
Glutamate-In the 60-h fasted human, gluconeogenesis occurs
(lo), and there is minimal decarboxylation of pyruvate (1).
The low rate of pyruvate decarboxylation is furtherevidenced
by the small percentage of 14Cin carbon5 compared to carbon
4 of glutamate (Table I). Carbons 1 and 2 of acetyl-coA are
the precursors of carbons 5 and 4 of a-ketoglutarate. There-
fore, there was minimal formation of [l-14C]acetyl-CoAfrom
the [2-I4C]acetate. Nevertheless, G1/G, ratios were <2 (1.5-
6988 Metabolism of Acetate

FIG. 1. Scheme proposed to ex-

plain distributions of 14C from [%
"'Clacetate in glucose and in gluta-
mate from glutamine conjugate of
phenyl acetate. [2-'4C]Acetate is me-
tabolized in the Krebs cycle in both liver
and muscle to form I4COsand labeled 01-
ketoglutarate. "CO, is fixed bycombi-
nation with pyruvatein liver to form
[''C]oxalacetate, which is converted in
liver into "C-labeled tu-ketoglutarate
and glucose. Glutamine conjugated to
phenylacetate is derived from the la-
beled wketoglutarate formed inboth
liver and muscle.

1.9) (Table 11). When the distributions are corrected for I4CO2 same site, as depicted in themodel, with isotopic equilibration
+
fixation, the ratios increase to near 5 (Table V), in keeping being extensive, the ((GI G2)/2)/G3 ratio obtainedwith [3-
with Krebs cycle metabolism and theoccurrence of gluconeo- 14C]lactate should then have been essentially the same as the
genesis. GJG3 ratio observed with[2-l4C]acetate.Theratios were
The G1/Gs ratios of near 2 that we haveobserved on markedly different. The ratios without correction for I4CO2
administering [2-I4C]acetate are similar to ratios other inves- fixation were 1.5-1.9 from [2-I4C]acetate (Table11), compared
tigators have found on administering carbon2 labeledacetate t o 5.1-7.4 from [3-14C]lactate (calculated from Table I1 of Ref.
or its equivalent. Ratios of between 1.6 and 3.0 were found in 1).Ratios were 4.8-7.8 in subjects given [3-14C]lactate after
blood glucose from normal animals and in glucose in urine an overnight fast(22).
from diabetic animals given [2-14C]acetate (9, 17, 18), [2-"C] 14C02 fixation contributed in a much greater proportion to
acetate (19), [2-14C]palmitate (17), and[6-14C]palmitate (20), the incorporations into carbons 3 and 4 of glucose when [2-
the 14C-labeled palmitates being converted to [2-14C]acetyl- l4C]acetate was substrate than when [3-I4C]lactatewas sub-
CoA in their oxidation. Ratios of 2.7-3.3 have been reported strate. This is apparent from the 3 times greater I4C specific
on administering [2-I4C]acetate to humans (10, 11).Hetenyi activity in urinary urea from acetate (Table 111) than from
et al. (8) gave 19 fasted rats[2-14C]acetate,and theaverage of lactate (Table IV of Ref. l ) , whereas the specific activity of
the ratios in glucose from 12 of the rats was 3.9. They used blood glucose was 3 timesgreater from lactate than from
that ratio to estimate carbon exchange in the Krebs cycle. acetate. This isevidence that acetate ismetabolized in a site
However, the ratios inglucose from the otherseven rats were other than lactate and that the site contributes in very signif-
not used because they were "unaccountably low, below 2.2." icant measure to the formation of 14C02.Fixation of I4CO2
The ratios of the distributions of I4C in glutamate (K2/K1) from [2-I4C]acetate has been assumed to be insignificant (7,
were also frequently <2 (1.3-2.3) (Table I), and as noted, Kz/ 9, 10). On correction for I4CO2fixation, the ratios in glucose
K1 would be expected to be 2 for a closed system. Also as observed with [2-14C]acetate (Table V) approached those ob-
noted, K4/Ky would be expected to be1rather than2-3 (Table served with [3-14C]lactate.Furthermore, when liverslices were
I). Similar distributions I were found incubated with [2-I4C]acetate (17, 23) and hepatocytes with
of label to those in Table
in glutamate from the liver of one rat given [2-'"C]acetate [2-"C]acetate (24) and the distribution of label in glucose was
(19) and rats given [2-14C]acetate(21). When the distributionsdetermined, the G1/G1 ratios were between 3.9 and 6.0, also
in glutamate in TableI were corrected for I4CO2fixation, the similar to the ratios obtained on giving [3-I4C]lactatein uiuo.
KS/K1 ratios increased to between 2.3 and 2.9 (Table IV). This isevidence that if [2-I4C]acetatewere metabolized solely
However, those ratios are still lower than the corrected GI/ in liver i n uiuo, the distributions in glucose would be similar
G:, ratios of 4.8-5.2 for glucose (Table V). In the 60-h fasted to those observed with [3-14C]lactate.
human, there is extensive equilibration between oxalacetate Rognstad (18)recently reported a comparison of the distri-
and fumarate (1). In this circumstance, if the glucose and bution in glucose on administering [2-I4C]acetate and[3-I4C]
glutamate were from a single oxalacetate pool, K2/K1 should lactate to rats. Since the G1/G3 ratio in glucose from acetate
have been similar to G1/GI3. was different from that from lactate, i.e. 2.5 as compared to
Comparisons of Distributions to Those for [3-14C]Lactate- 4.9, he concluded that the data obtainedusing [2-I4C]acetate
When [3-I4C]acetatewas given to 60-h fasted humans,I4C in are not applicable to the determinationof gluconeogenesis i n
carbon 1 of glucose was -1.2 times that in carbon 2. This vivo and that acetate was metabolized in a site other than
reflected the extensive equilibration between oxalacetate and liver. Our results are then in agreement with those conclu-
fumarate. A good approximation of what the G1/Gs ratio in sions. However, he gave acetate and lactate in bolus injections
glucose would be if isotopic equilibration were complete is and killed the rats 10min later.Underthoseconditions,
obtained by dividing G:rinto the average of G1 + G2, i.e. ((GI unless steady state was achieved or changes in the specific
+ G2)/2)/G:r (7). activity of pyruvate from lactate paralleled those from acetyl-
If [3-I4C]lactate and[2-"C]acetate were metabolized in the CoA formed from the acetate, the distributions from acetate
 Metabolism of Acetate 6989
and lactatewould probably have been different, even if acetate To the extent that the Krebs cycle functions as a "closed
and lactatewere metabolized at the same site. system" in muscle, glutamate formed from [2-14C]acetate
ExplanationforDistributions Obtainedwith [2-l4C1Ace- would be expected tohave about twice as much I4C in carbon
tate-The distributions of 14C in glucose then represent the 2 as in carbon 1,i.e. a K2/Kl ratio of 2. Glutamate formed in
sum o f t h e metabolism of [2-14C]acetate inone site,liver, and liver would be expected, with correction for l4CO2fixation, to
the fixation of 14C02 formed from the [2-14C]acetateprimarily have a K2/Kl ratio similar to theG1/G, ratio in glucose (-5)
in another site. The distributions in glucose cannot be ex- (Table V). Glutamatefromthe conjugate, formedfrom a
plained by acetate's conversion in tissues other thanliver t o mixture of the glutamatederived from liver and muscle, would
labeled glutamine and glutamate and then their conversion then in have a KJK1 ratio intermediate between 5 and 2, as it
liver to glucose. If the labeled glutamate was the precursor of does, i.e. 2.3-2.9 (Table IV).
glucose, the relative 14Cactivities of GI, GP, Gs, and G6 would In accord with our results, Kalderon et al. (27), on giving
be the average of Ks and K4, and G, and G4 the average of Kz [2-''C]acetate to rats, foundmuch less 13Cenrichment inliver
and Ks. Thus, for example, in subject U.E., where the percent glucose than in glutamate carbons. They concluded this was
I4Cdistributions in carbons 1-5 of glutamate were 10, 17, 17, incompatible witha pool of oxalacetate common to the Krebs
50, and 6 (Table I), respectively, the relative distributions cycle and gluconeogenesis. They offered, as an explanation,
would have been 34, 34, 12, 12, 34, and 34 in glucose carbons metabolic channeling of oxalacetate formed from pyruvate
1-6, respectively. G1/Gs would then have been2.8 rather than toward gluconeogenesis, resultingin incompletemixing of
1.8 (Table II).2 mitochondrial oxalacetate (Scheme 1 of Ref. 27). The K2/K1
Thedistributions in glutamatecannot be explained by and Kz/KB ratios in glutamate on administering [3-'4C]lactate
acetate's metabolism to glutamate in liver with fixation into should not thenhave been similar,as they were, to theG1/G:,
carbon 1 of the glutamate of 14C02 formed from the acetate and Gz/Gl ratios in glucose (1).This is because the carbons
in anothersite. If labeled glutamate, used in conjugation, were of oxalacetate, used in glucose formation, would not have
formedonly in liver,where glucose was formedand C 0 2 experienced as much randomization in the Krebs cycle as the
fixation occurred, 14C02fixation should have contributed ina carbons of oxalacetate used in a-ketoglutarate formation.
similar proportion to the14Cin carbon 1 of glutamate and in An alternative explanation for our results is that lactateis
carbons 3 and 4 of glucose. This was the case for [3-14C] converted to glucose and metabolized in the Krebscycle in a
lactate: an-30% contribution for both (l),but not for [2-"C] cellular site different from that where acetate is primarily
acetate; an -60% contribution for glucose; and a 30% contri- metabolized and gluconeogenesisdoes not occur, but that
bution for glutamate. This is reflected in similar ((G, + G2)/ both sites are in the liver. The metabolic picture in Fig. 1
2)/G, and K2/K1 ratios with [3-'4C]lactate, when corrected would be the same, except, rather than the peripheral site of
for I4CO2fixation (Tables VI and VI1 of Ref. l), but higher metabolism, that sitewould be in l i ~ e r . ~ . ~
GI/Ga than K2/K1 ratioswith [2-14C]acetate (Tables IV and Ethanol Metabolism-Since it seemed possible that if [2-
VI. 14C]acetate wasformed in theliver, distributions in glutamate
Incorporations of 14Cfrom the[2-14C]acetateinto glutamate and glucose maybetter reflectlivermetabolism, [2-l4Cc]
in peripheral tissuefs) and into glucose and glutamate in liver, ethanol, which is converted to [2-I4C]acetate liver
in (34), was
when combined with fixation of I4CO2by pyruvate in liver, administered to one subject. However, [2-14C]ethanol gave a
which 14C02formed in the oxidation of the [2-I4C]acetate, distribution similar to that of [2-14C]acetate(Tables I and 11),
provideapossible explanation for the distributions. Since in accord with the acetate,formed from ethanol, being metab-
acetate is extensively utilized and most glutamine is producedolized primarily by peripheral tissues (35-38).
by muscle (25, 26), muscleis depicted as the peripheral tissue [1,3-'4C]Acetone, formed from the [2-14C]acetate via [2,4-
(Fig. 1).When [2-"C]acetate was administered to rats,muscle 14C]acetoacetate, can be converted to[1,3-14C]lactate(39). Its
glutamate was labeled (27); and when [l-14C]acetate and [2- metabolism in the Krebs cycle would lower the K2/K1 ratio.
14
Clacetate were administered to rats, glutamate from carcass To test whether this pathway as well as 14C02fixation ac-
protein was labeled (21, 28)." counted for the incorporation into K1,one subject was given
' However, as noted after correction for I4CO2fixation, the G,/G, [l-14C]ethanol.[l-'4C]Acetyl-CoA,formedfrom [1-I4Cc]
ratio from [2-I4C]acetate approaches, butis less than, the ratio from ethanol, would form [2-14C]acetoneand then[2-14C]lactateby
[3-'4C]lactate. This differencecould be duetoincorporationinto
glucose of I4C from I4C-labeled glutamate formed in the periphery. Katz and Chaikoff (29) compared acetate's metabolism in the
Labeled lactate has alsobeen reported to be formed from [2-"C] intact animal and in liver slices. They concluded that in the intact
acetate (9). animal, acetate is oxidized mainly in extrahepatic tissues; and there-
, Whereas a contribution by kidney to the incorporation ofI4C fore, acetate's metabolism in uiuo may show little resemblance to its
from [2-I4C]acetate into blood glucose, as has been suggested (la), metabolism in isolated liver.
cannot be excluded, we believe liver is the primary source of the ' Consoli et al. (10, 11) concluded that using [2-14C]acetate,as
labeled glucose (Fig. 1). Thisis so, although kidneyslices were proposed by Katz (7), provides an accurate noninvasive measure of
reported to oxidize acetate more readily than liver slices and incor- gluconeogenesis. They concluded this because their estimates of the
poration of acetatecarboninto glucose as well as glutamate was rates of gluconeogenesis were those to be predicted from previous
appreciable (29). Our reasons include: 1) the large net production of measurements of splanchnic substrate balance and hepatic glycogen
glucose by liver, but, at most,only a very small production by kidney content. The ratesof gluconeogenesis were calculated by dividing the
in the 60-h fasted human (30); 2) the ratio G,/G:,= 2 in glucose from specific activities of glucose by the estimated specific activities of P-
diabeticanimals given [2-"C]palmitate (17) and[6-I4C]palmitate enolpyruvate. The specific activity of P-enolpyruvate was estimated
(20); and 3) the increases in concentration and specific activity of by multiplying half the specific activity of /3-hydroxybutyrate, as-
blood glucose in an overnight fasted subject we gave glucagon after sumed tobe the specific activity of hepatic mitochondrial acetyl-coA,
giving[2-"C]acetate and an oral glucose load. Concentrationin- by the ratio of the specific activity of P-enolpyruvate relative to that
creased from 3.6 to 5.2 mM and specific activity from 11.4 to 16.6, of acetyl-CoA, calculated from the G,/G:i ratio in glucose. An under-
presumably reflecting release of labeled glucose from liver of a higher estimation of the specific activity of the acetyl-coA,because half the
specific activity than that from the circulation. The percent distri- specific activity of the /3-hydroxybutyrate was less than that of the
bution of I4C in the glucose before glucagon was C, = 19.0, Cy = 14.6, acetyl-coA (31-33), compensated for by anoverestimation of the
C:I = 6.2, C, = 9.4, C , = 23.2, and C,; = 27.6 (97.3% recovery). After ratio of the specific activity of P-enolpyruvate to thatof the acetyl-
glucagon, it was C1 = 16.6, C, = 15.3, C:l = 8.9, C, = 13.4, C, = 22.9, CoA, because of a falsely low G,/G:{ratio due to"CO, fixation, could
and CI;= 22.9 (96.9% recovery), so G1/G:Iis -2. then have yielded the predicted rate of gluconeogenesis.
6990 Metabolism of Acetate
the pathway and hence a-[2,3-14C]ketoglutarate. Incorpora-
tion was only into carbons 1and 5 of the glutamate (Table I),
indicating that the pathway was not active and therefore did
not contribute to the incorporation of 14C into K1 from [2-
14C]acetate.
Acknowledgment-We thank Dr. Harland Wood for the very help-
ful suggestions he provided during the preparation of this paper.
REFERENCES
1. Magnusson, I., Schumann, W. C., Bartsch, G. E., Chandramouli,
V., Kumaran, K., Wahren, J., and Landau, B. R. (1991)J.Biol.
Chem. 266,6975-6984
2. Strisower, E. H., Kohler, G. D., and Chaikoff, I. L. (1952) J.Biol.
Chem. 198, 115-126
3. Weinman, E. O., Strisower, E. H., and Chaikoff, I. L. (1967)
Physiol. Reo. 37, 252-272
4. Goebel, R., Berman, M., and Foster, D. (1982) Fed. Proc. 41,96-
103
5. Hetenyi, G., Jr. (1982) Fed. Proc. 41, 104-109
6. Kelleher, J. K. (1986) Am. J. Physiol. 250, E296-E305
7. Katz, J. (1985)Am. J. Physiol. 248, R391-R399
8. Hetenyi G., Jr., and Ferrarotto, C., (1983) Biochem. Med. 29,
372-378
9. Hetenyi, G., Jr., Lussier, B., Ferrarotto, C., and Radziuk, J. (1982)
Can. J. Physiol. Pharrnacol. 60,1603-1609
10. Consoli A., Kennedy, F., Miles, J., and Gerich, J. (1987) J. Clin.
Znuest. 80, 1303-1310
11. Consoli, A., Nurjhan, N., Capani, F., and Gerich, J. (1989) Dia-
betes 38,550-557
12. Hellerstein, M.K., Kaempfer, S., Wu, K., Reid, S., and Shack-
leton, C. H. L. (1989) Clin. Res. 37, 130A (abstr.)
13. Radzuik, J. (1989)Am. J. Physiol. 257, E158-El69
14, Kadish, A. H., ~ i t l R.~ ,L.,and Sternberg, J. c. (1968) clin,
Chem. 14, 116-131
15. Magnusson, I., Chandramouli, V., Schumann, W. C., Kumaran,
K.9 Wahrent J., and Landau, B. R. (l987) J. CLin.Invest.
1748-1754
16. Bernstein, I. A., and Wood, H. G. (1957) Methods Enzymol. 4,
561-584
17. Kam, W., Kumaran, K., and Landau, B. R. (1978) J. Lipid Res.
19,591-600
18. Rognstad, R. (1988) Med. Sci. Res. 16, 293-294
19. Kalderon, B., Gopher, B., and Lapidot, A. (1987) FEBS Lett.
213,209-214
20. Abraham S., Chaikoff, I. L., and Hassid, W.D. (1952) J. Biol.
Chem. 195,567-581
21. Hill, R. J., Hobbs, D. C., and Koeppe, R. E. (1958) J. Biol. Chem.
230,169-178
22. Hostetler, K. Y.,Williams, H. R., Shreeve, W. W., and Landau,
B. R. (1969) J. Biol. Chem. 244,2075-2077
23. Antony, G. J., and Landau, B. R. (1968)J. Lipid Res. 9, 267-269
24. Petersen, K. F., Grunnet, N., Quistorff, B., Bock, K., and Tygs-
trup, N. (1988) Hepatology (Baltimore) 8, 1237 (abstr.)
25. Bulus, N.,Cersosimo, E., Ghishan, F., and Aburnrad, N. N. (1989)
Metab. Clin. Exp. 38, Suppl. 1, 1-5
26. Garber, A. J., Karl, I. E., and Kipnis, D. M. (1976) J. Biol. Chern.
25 1,836-843
27. Kalderon, B., Gopher, A., and Lapidot, A. (1987) FEBS Lett.
220,259-261
28. Koeppe, R. E., and Hill, R. J. (1955) J. Biol. Chem. 216, 813-
822
29. Katz, J., and Chaikoff, I. L. (1955) Biochim. Biophys. Acta 18,
87-101
30. Bjorkman, O., and Felig, P. (1982) Diabetes 31, 516-520
31.Des Rosiers, C., Montgomery, J. A., Garneau, M., David,F.,
Mamer, 0. A., Daloze, P., Toffolo, G., Cobelli, C., Landau, B.
R., and Brunengraber, H. (1990) Am. J. Physiol. 2 5 8 , E519-
E528
32. Fink, G., Desrochers, S., Des Rosiers, C., Garneau, M., David, F.,
Daloze, T., Landau, B. R., and Brunengraber, H. (1988) J. Biol.
Chem. 263,18036-18042
33. Norsten, C., and Cronholm, T. (1990) Biochem. J. 265,569-574
34. RLmLsy, C., and DemignL, D. (1983) Ann. Nutr. Metab. 27, 57-
70
35. Forsander, O., Raiha, N., and Suomalainen, H. (1960) Hoppe-
Seyler’s 2.Physiol. Chem. 318, 1-5
36. Jorfeldt, L., and Juhlin-Dannfelt, A. (1978)Metab. C h . EXP. 27,
97-106
37. Lindeneg, O., Mellemgaard, K., Fabricius, J., and Lundquist, F.
(1964) Clin. Sci. 2 7 , 427-435
38. Lundquist, F., Tygstrup, N., Winkler, K., Mellemgaard, K., and
Munck-Petersen, S. (1962) J. Clin. Invest. 41,955-961
39. Kosugi,K., Chandramouli, V., Kumaran, K., Schumann, W.C.,
and
R. Landau, B. (1986) J. Biol. Chem. 261,13179-13181