Brown adipose tissue triacylglycerol synthesis in rats
adapted to a high-protein, carbohydrate-free diet
M. N. BRITO, N. A. BRITO, S. R. C. BRITO, M. A. F. MOURA, N. H. KAWASHITA,
I. C. KETTELHUT, AND R. H. MIGLIORINI
Departments of Biochemistry and Physiology, School of Medicine, University of São Paulo,
Ribeirão Preto, 14049–900 São Paulo, Brazil
Brito, M. N., N. A. Brito, S. R. C. Brito, M. A. F. Moura,
N. H. Kawashita, I. C. Kettelhut, and R. H. Migliorini.
Brown adipose tissue triacylglycerol synthesis in rats adapted
to a high-protein, carbohydrate-free diet. Am. J. Physiol. 276
(Regulatory Integrative Comp. Physiol. 45): R1003–R1009,
1999.—Adaptation of rats to a high-protein, carbohydrate-
free (HP) diet induced a marked reduction of brown adipose
tissue (BAT) fatty acid (FA) synthesis from both 3H2O and
[14C]glucose in vivo, with pronounced decreases in the activi-
ties of four enzymes associated with lipogenesis: glucose-6-
phosphate dehydrogenase, malic enzyme, citrate lyase, and
acetyl-CoA carboxylase. In both HP-adapted and control rats,
in vivo incorporation of 3H2O and [14C]glucose into BAT
glyceride-glycerol was much higher than into FA. It could be
estimated that most of the glycerol synthetized was used to
esterify preformed FA. Glycerol synthesis from nonglucose
sources (glyceroneogenesis) was increased in BAT from HP
rats, as evidenced by an increased capacity of tissue frag-
ments to incorporate [1-14C]pyruvate into glycerol and by a
fourfold increase in the activity of phosphoenolpyruvate
carboxykinase activity, a key glyceroneogenic enzyme. The
data suggest that high rates of glyceroneogenesis and of
esterification of preformed FA in BAT from HP-adapted rats
are essential for preservation of tissue lipid stores, necessary
for heat generation when BAT is recruited in nonshivering
thermogenesis.
glyceroneogenesis; phosphoenolpyruvate carboxykinase; lipo-
genic enzymes
RATS ADAPTED TO A high-protein, carbohydrate-free (HP)
diet have been used for many years in our laboratory as
a model system to investigate adaptive mechanisms in
energy-linked metabolic processes. In previous studies
we have shown that despite a markedly reduced lipogen-
esis in both liver and adipose tissue, assessed in vivo by
the rate of incorporation of tritium from 3H2O into fatty
acids (FAs) (4, 27), rats adapted to an HP diet are able
to maintain considerable reserves of body fat. Thus
carcass FAs of rats fed the HP diet for 30 days amounted
to ⬃85–90% of values in control animals fed an isoca-
loric, balanced diet (27). We have subsequently shown
that the preservation of body fat stores in HP rats could
be due, at least in part, to a diet-induced reduction in
the activity of brown adipose tissue (BAT) (6), with a
consequent decrease in overall energy expenditure and
increase in metabolic efficiency. Indeed, BAT of rats
adapted to the HP diet has a reduced thermogenic
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0363-6119/99 $5.00 Copyright r 1999 the American Physiological Society
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capacity, as evidenced by a reduced response of inter-
scapular BAT (IBAT) temperature to norepinephrine
infusion and by decreases in IBAT weight, mitochon-
drial protein, cytochrome oxidase activity, and mitochon-
drial binding of GDP (6). These signs of reduced BAT
thermogenesis in HP-adapted rats were accompanied
by a reduction in lipogenic activity, estimated in vivo by
the incorporation of 3H2O into tissue FAs (6). The
present experiments were undertaken to further inves-
tigate the diet-induced alterations in BAT lipogenesis.
Thus the effects of the HP diet on the activity of
enzymes involved in lipid synthesis and on the relative
contribution of glucose carbon and carbon from other
sources to the in vivo synthesis of FA and glycerol
moieties of BAT triacylglycerols (TAGs) are here re-
ported. In the course of these experiments it was found
that both in control and HP rats the rate of label
incorporation into glyceride-glycerol was several times
higher than into FAs. This prompted us, especially in
view of the lack of carbohydrate in the diet of HP rats,
to examine more closely BAT glyceroneogenesis, a
process that has been little investigated in this tissue.
To this end, the rate of in vitro incorporation of several
labeled glyceroneogenic substrates into glyceride-
glycerol, as well as the activity of phosphoenolpyruvate
carboxykinase, a key glyceroneogenic enzyme, were
determined in IBAT from control and HP-adapted rats.
MATERIALS AND METHODS
Animals and Treatment
Male Wistar rats weighing initially 60–90 g were housed in
suspended, wire-bottom cages, with water ad libitum, in a
room kept at 25 ⫾ 2°C with a 12:12-h light-dark cycle. The
animals were adapted for 20 days to an HP-purified diet
containing 70% casein, no carbohydrate, and 8% corn oil or to
a balanced, control diet, containing 17% casein, 66% carbohy-
drate, and 8% corn oil. The two diets, which were approxi-
mately isocaloric and contained equal amounts of vitamins
and minerals, have been described in detail (6). As in previous
studies with the same diet, after an initial period of adapta-
tion of a few days, food ingestion and the rate of body weight
increase were similar for the two groups of rats (17). The
animals weighed 180–200 g when used for the experiments,
which were performed between 8:00 and 10:00 AM.
In Vivo Lipogenesis Studies
Experimental approach. The glucose contribution to the
synthesis of glycerol and fatty acid moieties of TAG was
evaluated by determining simultaneously in the same animal
the rate of incorporation from tritiated water, which esti-
mates total synthesis (from all carbon sources), and of 14C
from glucose into two TAG moieties of IBAT. The assumptions
and supportive arguments for the adequacy of 3H2O for
R1003
R1004 HIGH-PROTEIN DIET AND TRIACYLGLYCEROL SYNTHESIS IN BAT
measurement of lipid synthesis from all carbon sources have
been presented by Windmueller and Spaeth (31) and Jungas
(15). The flux of glucose carbon to IBAT FA or glycerol was
estimated using the semicompartmental approach described
by Baker and Huebotter (2), which is a modification of the
noncompartmental approach of Shipley et al. (29) and com-
bines features of both noncompartmental and compartmental
analyses. The semicompartmental analysis requires measure-
ment of the specific activity-time curve of the precursor after
a single injection of a radioactive tracer [as in the method of
Shipley et al. (29)] and the measurement of the radioactivity
in an ‘‘end product’’ at any point in time (60 min in the present
study). The technique’s assumptions and supportive argu-
ments are described in Ref. 2. It was assumed that no
appreciable turnover of 3H- or 14C-labeled product occurred
during the experimental period, so the rates obtained are
minimal values.
Label injection and isolation of tissue TAG-FAs and glyc-
erol. [U-14C]glucose (10 µCi) and 3H2O (5 mCi) dissolved in 0.5
ml saline were injected into fed, nonanesthetized rats through
a Silastic (Dow Corning, Midland, MI) catheter inserted into
the right jugular vein 2 days before the experiment. After the
catheter was flushed with saline, and with the rat free in its
cage, blood samples of 0.2 ml were taken 1, 5, 15, 30, and 60
min after labels injection for determination of [14C]glucose
specific activity. Immediately after obtainment of the 60-min
sample, which was also used for determination of plasma
water specific activity, the animals were killed by cervical
dislocation. The IBAT was rapidly removed, carefully cleaned
free of adhering fat and muscle, and weighed. IBAT total
lipids were extracted with 2:1 chloroform-methanol by the
procedure of Folch et al. (9). 3H2O was removed from the lower
phase (predominantly chloroform) by washing three times
with an upper phase mixture (9). After each shaking, the
tubes were briefly centrifuged to sharpen the phase boundary
and the upper phase was aspirated and discarded. The
chloroform phase was evaporated to dryness under N2, and
the TAGs were hydrolyzed with ethanolic KOH for 1 h at
70°C. After extraction of nonsaponifiable lipids and acidifica-
tion with 6 N H2SO4, the 14C- and 3H-labeled FA was
extracted with petroleum ether, and the extract was evapo-
rated to dryness in a scintillation vial and dissolved in
toluene-2,5-diphenyloxazole (PPO). A volume of the aqueous
hydrolysate containing 14C- and 3H-labeled glycerol was
dissolved in toluene-triton-PPO-POPOP.
Determination of plasma glucose and body water specific
radioactivity. Plasma [14C]glucose was isolated by thin-layer
chromatography and its radioactivity measured as described
by Baker et al. (3). The concentration of plasma glucose was
determined with glucose oxidase in a Beckman (Fullerton,
CA) glucose analyzer. Body water specific activity was deter-
mined directly on aliquots of diluted (20 times) plasma
dissolved in toluene-triton-PPO-POPOP.
Radioactivity measurements. The degree of quenching in
each sample was obtained to enable calculation of radioactiv-
ity in disintegrations per minute. Simultaneous liquid scintil-
lation counting (LS 7600 Beckman spectrometer) of the 3H
and 14C content of FA or glycerol was performed using a
channels ratio method (12).
Calculations. Plasma glucose levels of HP and control rats
1 min postinjection were (in mg/dl) 117 ⫾ 3 (6 rats) and 125 ⫾
4 (6 rats), respectively. In the two groups, plasma glucose
concentration of each animal did not change significantly
during the experimental period (data not shown), a require-
ment of the technique used. The curves of plasma glucose
specific activity were fitted to two terms exponential equa-
tions, whose parameters were used in the calculations (2). For
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the calculations of lipid synthesis in the experiments with
3H O, it was assumed that the specific activity of intracellular
2
water was identical to that of plasma water. Rates of lipid
synthesis were calculated assuming that each glycerol and
each FA incorporated into TAG contained 3.3 and 13.3 atoms
of tritium, respectively (15, 31).
In Vitro Experiments
The rats were killed by cervical dislocation. The IBAT was
removed, cleaned free of fat and muscle, and cut into small
pieces of ⬃5 mg. Portions of 100 mg were incubated in 5 ml of
Krebs-Henseleit bicarbonate buffer, pH 7.4, in which appropri-
ate substrates were dissolved, as indicated in RESULTS. Incuba-
tion was carried out at 37°C with constant shaking for 2 h.
The tissue fragments were then rinsed three times with 0.9%
NaCl and transferred to 2:1 chloroform-methanol. The proce-
dure used for isolation and determination of 14C in final
products was the same as that described for the in vivo
experiments.
Measurement of Enzyme Activities
For determination of glucose-6-phosphate dehydrogenase
(G6PDH), malic enzyme (ME), and citrate lyase (CLY) activ-
ity, IBAT was homogenized in ice-cold 10 mM Tris buffer, pH
7.4, containing 0.32 M sucrose, 2 mM EDTA, and 5 mM
mercaptoethanol. After centrifugation at 10,000 g for 10 min
and removal of the top fat layer, the supernatant was
centrifuged for 1 h at 100,000 g to obtain a new supernatant,
which was used to determine the activity of the enzymes.
G6PDH was assayed as described by Lee (18), and ME was
assayed by the method of Ochoa (23), with the modifications
proposed by Hsu and Lardy (14). Both assays were performed
by measuring the rate of formation of NADPH. In addition to
adequate amounts of the 100,000 g supernatant, the assay
mixture contained, for G6PDH, 0.1 M Tris, pH 8.0, 1 mM
NADP⫹, and 1 mM D-glucose-6-phosphate and, for ME, 67
mM triethanolamine, pH 7.4, 5 mM L-malate, 4 mM MnCl2,
and 0.2 mM NADP⫹. CLY was assayed as described by Srere
(30), measuring the rate of oxidation of NADH in a assay
mixture containing 50 mM triethanolamine, pH 7.7, 7 mM
ATP-Mg, 10 mM potassium citrate, 0.1 mM NADH, 10 mM
mercaptoethanol, 0.24 mM CoA, NAD-malate dehydrogenase
(1 unit/ml), and aliquots of the 100,000 g supernatant.
Phosphoenolpyruvate carboxykinase was assayed by the
method of Chang and Lane (7) in 100,000 g supernatants
obtained after homogenization of IBAT in 20 mM triethanol-
amine buffer, pH 7.5, containing 0.2 M sucrose, 5 mM
mercaptoethanol, and 1 mM EDTA. The incorporation of
[14C]bicarbonate (2 µCi, Amersham) into acid-stable product
was determined in an assay mixture (final pH 6.9–7.1)
containing 100 mM imidazole, pH 6.6, 2 mM MnCl2, 1 mM
glutathione (GSH), 1.25 mM IDP, 2.5 mM NADH, 50 mM
KHCO3, 1.5 mM phosphoenolpyruvate, malate dehydroge-
nase (2 units/ml), and supernatant. For determination of
acetyl-CoA carboxylase activity, IBAT was homogenized in
50 mM potassium phosphate buffer, pH 7.3, containing 2 mM
EDTA, 4 mM GSH, and albumin (10 mg/ml). The assay was
carried out as described by Halestrap and Denton (11) by
measuring the incorporation of [14C]bicarbonate (3 µCi) into
acid-stable material after incubation of 1,500 g supernatants
of whole homogenate with citrate. The assay mixture con-
tained 100 mM Tris · HCl, pH 7.4, 5 mM ATP, 10 mM MgCl2, 1
mM GSH, 15 mM NaHCO3, 0.15 mM acetyl-CoA, and 5 mg/ml
albumin. Protein concentration was determined as described
by Lowry et al. (19).
 HIGH-PROTEIN DIET AND TRIACYLGLYCEROL SYNTHESIS IN BAT
Statistical Methods
Differences between groups were analyzed using ANOVA,
with P ⬍ 0.05 as the criterion of significance.
RESULTS
In Vivo Lipogenesis
The data in Table 1 show that adaptation to the HP
diet resulted in a marked reduction in the rate of
incorporation of 3H from tritiated water into IBAT
TAG-FAs. In both HP and control rats the rate of
incorporation of [14C]glucose into BAT TAG-FA was a
small fraction of the rate obtained with 3H2O, which
estimates synthesis from all carbon sources. Also, incor-
poration rate of hexose carbon into IBAT TAG-FA of
rats adapted to the HP diet was reduced to ⬃25% of
that in rats fed the balanced diet. In both experimental
groups the rates of incorporation of 3H2O or [14C]glucose
into IBAT TAG-glycerol were much higher than into
tissue TAG-FAs (Table 1). In contrast to the reduction
in FA synthesis, adaptation to the diet did not affect
TAG-glycerol synthesis from either 3H2O or [14C]glu-
cose. However, the proportion of label incorporated into
IBAT TAG-glycerol was much higher in HP-adapted
rats than in controls. Thus, whereas in control animals
the ratio of label incorporation into glycerol over incor-
poration into fatty acid was ⬃3 for both 3H and 14C, in
HP rats this ratio was 8 and 13 for 3H and 14C,
respectively.
Lipogenic Enzymes
In agreement with the reduction in BAT TAG-FA
synthesis induced by the HP diet, the activity of four
enzymes associated with FA synthesis was markedly
reduced in IBAT from rats adapted to the HP diet (Fig.
1). The activity of ME and of CLY was reduced to only
8% of values in animals fed the control diet. G6PDH
and acetyl-CoA carboxylase activities were reduced to
18 and 33%, respectively, of control values.
In Vitro Experiments With Glyceroneogenic Substrates
The results obtained by incubating fragments of
IBAT with several concentrations of [2-14C]pyruvate
are shown in Fig. 2. The rate of incorporation of
[2-14C]pyruvate into TAG-FA was directly related to the
substrate concentration in both HP and control rats,
Table 1. In vivo incorporation of 3H2O and
[U-14C]glucose into glyceride-fatty acid and glycerol
of IBAT from rats adapted to HP or control diet
Glyceride-Fatty Acid Glyceride-Glycerol
Control HP diet Control HP diet
3H O
2 37.8 ⫾ 2.3 15.0 ⫾ 1.4* 118.8 ⫾ 24.4 115.4 ⫾ 16.3
(n ⫽ 10) (n ⫽ 14) (n ⫽ 10) (n ⫽ 14)
[U-14C]glucose 7.0 ⫾ 1.2 1.8 ⫾ 0.2† 22.3 ⫾ 4.8 22.9 ⫾ 8.7
(n ⫽ 9) (n ⫽ 9) (n ⫽ 10) (n ⫽ 15)
Values are means ⫾ SE in nmol · g⫺1 · min⫺1; n ⫽ no. of rats. IBAT,
interscapular brown adipose tissue; HP, high protein, carbohydrate
free. * P ⬍ 0.05 vs. control. † P ⬍ 0.01 vs. control.
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R1005
Fig. 1. Activity of glucose-6-phosphate dehydrogenase (G6PDH),
malic enzyme (ME), citrate lyase (CLY), and acetyl-CoA carboxylase
(ACCX) in interscapular brown adipose tissue (IBAT) from rats
adapted to high-protein, carbohydrate-free (HP) or control diet. * P ⬍
0.05. ** P ⬍ 0.01.
but was always smaller in IBAT from HP rats (Fig. 2A).
On the other hand, the rate of [2-14C]pyruvate incorpo-
ration into TAG-glycerol was not significantly affected
by the diet at any of the concentrations of substrate
used (Fig. 2B). The proportion of 14C from pyruvate
incorporated into TAG-glycerol, expressed in Fig. 2C as
percentage of total label incorporation into TAGs
[[14C]glycerol ⫻ 100/([14C]glycerol ⫹ [14C]FA)] was much
higher in IBAT from HP rats. Figures 3 and 4 show the
results of experiments similar to those carried out with
[2-14C]pyruvate, but using as substrates several concen-
trations of [U-14C]lactate (Fig. 3) or [U-14C]alanine (Fig.
4). Although the rate of incorporation of 14C from these
two substrates, especially from alanine, into BAT TAG
was lower than that obtained with pyruvate, the effects
of the HP diet were qualitatively similar to those of the
experiments with [2-14C]pyruvate. Thus adaptation to
the HP diet induced a reduction in IBAT TAG-FA
synthesis from both lactate (Fig. 3A) and alanine (Fig.
4A) and an increase in the proportion of these metabo-
lites incorporated into TAG-glycerol (Figs. 3C and 4C).
Table 2 show the results of experiments in which the
rate of incorporation of carbon-1 from pyruvate into
IBAT glyceride-glycerol was compared with the rate of
incorporation of carbon-2. As anticipated, incorporation
of [1-14C]pyruvate into TAG-FA acids was negligible
(data not shown), because carbon-1 is lost in the
formation of acetyl-CoA. In IBAT from both HP-
adapted and control rats, the rate of incorporation of
[1-14C]pyruvate into glycerol was ⬃40–50% of rates
obtained with [2-14C]pyruvate (Table 2). These data are
consistent with the presence of a functioning dicarbox-
ylic acid shuttle in BAT. In the experiments with
[1-14C]pyruvate, when label incorporation into FA was
negligible, incorporation into glyceride-glycerol was
significantly higher in IBAT from HP-adapted rats
than in controls in tissues incubated with 0.2 and 1.0
mM of [1-14C]pyruvate (Table 2). With 5.0 mM pyru-
vate, statistical significance was not attained, but there
was a clear tendency for higher values (P ⬍ 0.10) in
HP-fed rats (Table 2).
R1006 HIGH-PROTEIN DIET AND TRIACYLGLYCEROL SYNTHESIS IN BAT

that glucose does not constitute a preferential sub-

strate for FA formation in BAT but is a major contribu-
tor to glyceride-glycerol synthesis, similar to what
has been found for white adipose tissue (10, 16, 24).
Indeed, even in rats fed the balanced, carbohydrate-
rich diet, TAG-FA synthesis from glucose constituted
only ⬃18% of IBAT total FA synthesis, while the rate of
hexose incorporation into glyceride-glycerol was three
times higher than that into FA (Table 1). It can also be
inferred from these experiments that most of the
glyceride-glycerol synthesized by BAT is utilized for
esterification of preformed FA, which include, in addi-
tion to FAs recycled after hydrolysis of stored TAGs,
FAs taken up by the tissue from the circulation (FA

Fig. 2. In vitro incorporation of [2-14C]pyruvate at concentrations of

0.2, 1.0, and 5.0 mM into triacylglycerol (TAG)-fatty acids (FAs) (A)
and into glyceride-glycerol (B) of IBAT from rats adapted to HP or
control diet. C: percentage of total label incorporation into TAG
incorporated into TAG-glycerol (see text). * P ⬍ 0.05. ** P ⬍ 0.01.

Phosphoenolpyruvate Carboxykinase Activity

In agreement with the data above, indicative of an
increased BAT glyceroneogenesis in HP-adapted rats,
the activity of phosphoenolpyruvate carboxykinase ac-
tivity (in nmol · mg protein⫺1 · min⫺1 ) was about four
times higher in IBAT from these animals (28.4 ⫾ 4.2, 7
rats) than in controls fed the balanced diet (6.8 ⫾ 0.6, 9
rats).
DISCUSSION
Fig. 3. In vitro incorporation of [U-14C]lactate at concentrations of
0.2, 1.0, and 5.0 mM into TAG-FAs (A) and into glyceride-glycerol (B)
Before discussing the changes in lipogenic activity of IBAT from rats adapted to HP or control diet. C: percentage of total
induced by the HP diet, it is interesting to note that the label incorporation into TAG incorporated into TAG-glycerol (see
results of the in vivo experiments (Table 1) indicate text). * P ⬍ 0.05. ** P ⬍ 0.01.

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 HIGH-PROTEIN DIET AND TRIACYLGLYCEROL SYNTHESIS IN BAT
Fig. 4. In vitro incorporation of [U-14C]alanine at concentrations of
0.2, 1.0, and 5.0 mM into TAG-FAs (A) and into glyceride-glycerol (B)
of IBAT from rats adapted to HP or control diet. C: percentage of total
label incorporation into TAG incorporated into TAG-glycerol (see
text). * P ⬍ 0.05. ** P ⬍ 0.01.
produced by breakdown of lipoproteins and albumin-
bound free FAs). In fact, the portion of glycerol that is
utilized for esterification of preformed FA can be ob-
tained by discounting the glycerol used to esterify FA
synthesized de novo, which corresponds to one-third of
the glyceride-FAs synthesized from 3H2O (Table 1), if it
is assumed that 3 mol of FA are esterified by 1 mol of
glycerol. Thus it can be estimated [([3H]glycerol ⫺
1 [3H]FA) ⫻ 100/[3H]glycerol] that ⬃89% of total glyc-
⁄3
erol synthesized in BAT of rats fed the balanced diet
was utilized to esterify preformed FA. For HP-fed rats
this percentage is even higher: 96%.
We have previously reported that BAT of rats adapted
to the HP diet has a reduced thermogenic capacity that
is accompanied by a decreased lipogenic activity, esti-
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R1007
mated by the incorporation of 3H2O into tissue FAs (6).
The present data show that FA synthesis from glucose
in vivo is also reduced and that the activities of G6PDH
and ME, generators of NADPH for lipid synthesis, as
well as of CLY and acetyl-CoA carboxylase, which
participate in the FA synthesis pathway, are markedly
reduced in BAT from rats fed the HP diet. The available
evidence suggests that the marked reduction in BAT
lipogenesis in HP-adapted rats is probably due to a
combination of neural and hormonal factors. Numerous
studies, reviewed by Himms-Hagen (13), suggest that
chemical signs elicited by qualitative and quantitative
changes in the diet modulate the activity of sympa-
thetic neurons in the ventromedial and other hypotha-
lamic areas that control BAT thermogenesis. It has
been found that electrical stimulation of the ventrome-
dial hypothalamus markedly increases lipid synthesis
in BAT (28) and that this effect is almost completely
abolished after sympathetic denervation of the tissue
(22). We have recently shown (5) that norepinephrine
turnover rate, which is mainly dependent on sympa-
thetic impulse traffic, is greatly reduced in BAT from
HP-fed rats, suggesting that the decrease in lipogenesis
may be due to a hypothalamic-mediated suppression of
BAT sympathetic activity. On the other hand, rats fed
high-protein diets have low levels of circulating insulin
and high levels of glucagon (8, 25), and it has been
found that insulin stimulates BAT lipogenesis, both in
vivo and in brown adipocytes in vitro (20, 21, 26),
raising the possibility that a low insulin-to-glucagon
ratio at the tissue level contributes to the reduction in
FA synthesis. Plasma insulin levels in rats of the
present work, determined by radioimmunoassay, were
(in µU/ml) 29 ⫾ 5 for HP (5 animals) and 46 ⫾ 8 for
controls (5 animals). Further experiments are needed
to determine the relative importance of neural and
hormonal factors.
The data of the present work indicate that glyceroneo-
genesis is very active in BAT, enabling the tissue to
synthesize glycerol from nonglucose sources by forming
phosphoenolpyruvate from pyruvate via the dicarbox-
ylic shuttle and subsequent production of glycerol
phosphate by a partial reversal of glycolysis. The data
in Table 1 show that even in rats fed the balanced diet,
glycerol synthesis from noncarbohydrate sources, esti-
mated by subtracting synthesis of glycerol from [14C]glu-
cose from the rates obtained with 3H2O, represented
Table 2. In vitro incorporation of [1-14C]pyruvate and
[2-14C]pyruvate at concentrations of 0.2, 1.0, and 5.0
mM into glyceride-glycerol by fragments of IBAT from
rats adapted to HP or control diet
[1-14C]Pyruvate [2-14C]Pyruvate
Pyruvate,
mM Control HP diet Control HP diet
0.2 3.1 ⫾ 0.6 6.6 ⫾ 0.8† 10.8 ⫾ 1.8 11.4 ⫾ 4.3
1.0 20.0 ⫾ 2.8 30.3 ⫾ 3.4* 58.0 ⫾ 8.9 85.0 ⫾ 14.9
5.0 99.7 ⫾ 11.1 132.9 ⫾ 11.5 244.0 ⫾ 31.3 227.8 ⫾ 25.4
Values are means ⫾ SE in nmol · g⫺1 · min⫺1 of 6 rats. * P ⬍ 0.05 vs.
control. † P ⬍ 0.01 vs. control.
R1008 HIGH-PROTEIN DIET AND TRIACYLGLYCEROL SYNTHESIS IN BAT
⬃81% of total glycerol production. The present study
also shows that the glyceroneogenic activity is in-
creased in BAT from rats adapted to the HP diet, as
evidenced by the markedly higher proportion of
[2-14C]pyruvate, [14C]lactate, and [14C]alanine incorpo-
rated into glycerol compared with FA, by the increased
conversion of [1-14C]pyruvate into glycerol, as well as
by the fourfold increase in the activity of BAT phospho-
enolpyruvate carboxykinase, an enzyme that has been
shown to play a regulatory role in white adipose tissue
glyceroneogenesis.
The results of the present work emphasize the impor-
tance of an active production of glycerol phosphate for
the normal functioning of BAT. A significant part of this
production seems to be effected through glyceroneogen-
esis, which proceeds at high rates even in animals fed a
balanced, carbohydrate-rich diet. Only a relatively
small part of the glycerol phosphate produced appears
to be used to esterify newly synthesized FAs, the
majority being directed to esterification of preformed
fatty acids. Because, in contrast to white adipose
tissue, BAT has an appreciable glycerokinase activity,
it seems reasonable to conclude that a considerable
part of these preformed FAs were FAs taken up from
the circulation, especially, in the fed state, FA produced
by hydrolysis of chylomicrons. High rates of glycerol
synthesis and of uptake and esterification of preformed
FAs seem essential to ensure adequate stores of TAGs,
necessary for a normal BAT thermogenic activity. In-
deed, activation of heat production by BAT in both
diet-induced and nonshivering thermogenesis is associ-
ated with stimulation of tissue TAG hydrolysis to
produce FAs, which are both substrates and uncoupling
messengers for BAT mitochondria (13).
The control of BAT metabolism is well illustrated by
the adaptive changes that occur in rats adapted to the
HP diet. We found in a previous work (6) that despite
the extremely reduced FA synthesis, the lipid content
(mostly TAGs) of IBAT from rats fed the HP diet for
20–30 days amounted to 53% (per whole tissue), or 87%
(per gram tissue) of values in animals fed the control
diet. This capacity of BAT from HP-adapted rats to
maintain substantial reserves of lipid can be explained
by the high rate of glyceroneogenesis and esterification
of preformed fatty acids suggested by the present data.
Lipid stores are necessary for an adequate response of
BAT when the tissue is recruited in nonshivering
thermogenesis. We have recently shown (5) that the
depressed sympathetic activity in IBAT from HP rats is
rapidly restored after acute cold exposure (4°C), with
no change in the body (colonic) temperature, despite
the initially reduced thermogenic capacity. There can
be little doubt that the lipid stored in BAT from HP rats
was essential for heat production by sympathetic activa-
tion. The results of the cold exposure experiments (5)
also illustrate the prevalence of heat generation for
thermal protection in a situation where BAT thermogen-
esis is reduced to increase the metabolic efficiency of
the diet.
We thank Maria A. R. Garófalo, Neusa M. Z. Resano, and José R.
Oliveira for technical assitance.
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Copyright © 1999 the American Physiological Society. All rights reserved.
This work was supported by grants from the Fundação de Amparo
à Pesquisa do Estado de São Paulo (FAPESP 97/0974–0) and from the
Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq
510923/93–3).
Present address of M. N. Brito and N. A. Brito: Dept. of Morpho-
physiological Sciences, State University of Maringá, 87020–900
Maringá-PR, Brazil.
Address for reprint requests and other correspondence: R. H.
Migliorini, Dept. of Biochemistry, School of Medicine-USP, 14049–
900 Ribeirõ Preto-SP, Brazil. (E-mail: rhmiglio@fmrp.usp.br).
Received 3 August 1998; accepted in final form 4 December 1998.
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Copyright © 1999 the American Physiological Society. All rights reserved.