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Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil
Address all correspondence and requests for reprints to: Antonio C. Bianco, M.D., Ph.D., Departamento de Fisiologia e Biofísica, ICB-USP, Avenida Prof. Lineu Prestes 1524, 05508–900 Sao Paulo, Sao Paulo, Brazil. E-mail: acbianco{at}usp.br
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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40%) when no additions were made to the
incubation medium. T4 or T3 partially prevented
that inhibition at physiological concentrations (65 x
10-9 and 0.77 x 10-9 M,
respectively), whereas a receptor-saturating concentration of
T3 (154 x 10-9 M) doubled
the lipogenesis rate. The addition of 10-6 M
NE inhibited lipogenesis acutely (50% by 12 h) and was
followed by a progressive stimulation that reached 2-fold by 48
h, but only in the presence of T4. Furthermore, NE did not
attenuate T3 (154 x 10-9
M)-induced lipogenesis. Both the inhibition and the
stimulation of lipogenesis caused by NE showed a strong dose-response
relationship within the range of 10-11-10-5
M. The role of local 5'-DII was further tested by
incubating brown adipocytes with 10-6 M NE and
T4 (65 x 10-9 M) in the
presence of 100 µM iopanoic acid, a potent inhibitor of
5'-DII. Although iopanoic acid did not affect the T3
stimulation of lipogenesis, it did block the 2-fold stimulation of
lipogenesis triggered by NE in the presence of T4,
confirming the mediation of 5'-DII in this process. In conclusion,
lipogenesis in brown adipose tissue is under complex hormonal control,
with key roles played by NE, thyroid hormones, and local 5'-DII. As in
other tissues, NE-generated signals acutely (12 h) inhibited
lipogenesis. However, the presence of the 5'-DII generated enough
T3 to stimulate lipogenesis and gradually reverse the
short-lived NE-induced inhibition, leading to the 2- to 3-fold response
observed at later time points. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Norepinephrine (NE) released by the profuse sympathetic nervous system
(SNS) innervation triggers most of the BAT biochemical pathways,
eventually leading to heat liberation. In addition to modulating
several processes that are primarily dependent on the cAMP/protein
kinase A (PKA) pathway, NE causes a several-fold stimulation of type II
5'-deiodinase (5'-DII) (4), an enzyme that produces
T3 from T4 within the brown
adipocytes. Shortly after cold exposure is initiated, the occupancy of
BAT nuclear T3 receptors is enhanced from its
baseline of 70% up to virtual saturation (5). As a result,
processes that are primarily dependent on thyroid hormone are also
activated. Therefore, the physiological changes that take place during
cold/NE stimulation of BAT are actually a composite of interactions
between NE- and thyroid hormone-generated signals that eventually lead
to heat liberation. In fact, the role of local 5'-DII appears to be so
important for BAT physiology that, by being increased several-fold
during hypothyroidism, the local generation of T3
reduces the effects of hypothyroxinemia on BAT nuclear
T3 receptor occupancy (6).
We have shown that locally produced T3 interacts
with NE-dependent signals to stimulate uncoupling protein (UCP) gene
expression, which is the limiting mitochondrial protein for BAT
thermogenesis (7, 8, 9). Furthermore, data obtained in vivo
(10, 11, 12) indicate that the SNS stimulation of BAT during cold exposure
increases the activity and/or expression of key lipogenic enzymes,
i.e. malic enzyme (ME), glucose-6-phosphate dehydrogenase,
acetyl coenzyme A (CoA) carboxylase (ACC), as well as tissue
lipogenesis measured by 3H2O incorporation (13, 14). In addition, hemidenervation of the interscapular BAT of rats
maintained at thermoneutrality resulted in a rapid 30–40% drop in
ME activity (12).
It is intriguing, however, that the SNS induces lipogenesis in BAT, the opposite of what NE causes in liver and lactating mammary gland, the other two important lipogenic tissues (15, 16, 17). In studies involving differentiating brown adipocytes (18, 19), where the role of local 5'-DII was not accounted for, NE and other inducers of the cAMP pathway, such as glucagon and forskolin, all caused moderate to complete inhibition of S14 gene expression, a nuclear protein closely linked to tissue lipogenesis (20). This would, in turn, suggest an indirect effect(s) in SNS-induced BAT lipogenesis. In previous publications (10, 12), we tentatively explained the apparent discrepancy of NE modulation of BAT lipogenesis by the local generation of T3. Our in vivo data suggest that the T3-induced stimulation of lipogenesis may overcome the inhibition directly caused by NE. Nevertheless, in addition to providing only a tentative explanation, the in vivo model has limitations in terms of distinguishing direct from indirect effects, which in the case of lipogenic enzymes are particularly important given their multifactorial regulation.
The aim of the present study was to investigate the mechanisms involved in the hormonal regulation of 1) de novo lipogenesis as estimated by 3H2O incorporation and 2) the activities of ME and ACC in freshly dispersed brown adipocytes, particularly the individual roles of NE and thyroid hormones. ME is an NADPH-generating enzyme whose activity positively correlates with the rate of de novo fatty acid biosynthesis and that of ACC, which is, in turn, the rate-limiting enzyme for this pathway (10, 21).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Unless otherwise specified, all drugs and reagents were purchased from Merck (Darmstadt, Germany) or Sigma Chemical Co. (St. Louis, MO). After the animals were killed, the interscapular and cervical BAT pads were rapidly dissected and placed in basic medium kept at room temperature. This was DMEM buffered with 44 mM sodium bicarbonate and 20 mM HEPES, pH 7.4. On the morning of each experiment the medium was supplemented with 4% fatty acid-free BSA (final concentration). After being freed from visible white fat and minced with scissors, the tissue was suspended in 1 ml medium/rat containing 2 mg/ml collagenase (Worthington Biochemical Corp., Freehold, NJ). Digestion was carried out at 37 C in a Nalgene (Nalge Company, Rochester, NY) bottle gassed with 5% CO2-95% O2 and incubated in a Dubnoff shaker set at 140 cycles/min. After 10 min, the cells were harvested on a chiffon nylon membrane, transferred to a clean Nalgene bottle, and returned to the incubator for an additional 20–25 min. The end of the digestion time was determined by the appearance of homogeneous turbidity in the medium and the disappearance of most visible fragments. The digestion was stopped by the addition of 1 vol collagenase-free medium. The cells were then filtered into plastic graduated centrifuge tubes and left to float. The undigested fragments were discarded. The infranatant was carefully aspirated with a glass micropipette, and the cells were resuspended with new collagenase-free medium. The procedure was repeated two or three times. Finally, the cells were transferred to a clean Nalgene bottle and counted under the microscope. The yield was usually 5–6 x 106 cells/rat.
In any given experiment, cells obtained from BAT pads of 20–25 rats were pooled and then separated into different 2.0-ml Eppendorf tubes containing culture medium down to a final volume of 1.5 ml at a concentration of 1 x 106/ml. The caps of all tubes were perforated with a 30-gauge disposable needle to allow ventilation during incubation. Depending on the experiment, the incubations lasted 2–48 h and were carried out in a cell culture incubator with a water-saturated atmosphere containing 5% CO2 and 95% O2.
At the end of the incubation period, cell viability was regularly
estimated by trypan blue exclusion and was always greater than 95%.
Another indication that the cells remained alive during the many hours
of incubation was that in some experiments when the amount of DNA of
paired tubes was measured at the beginning and the end of the
incubation period, the differences were never greater than 10% of the
initial amount. During pilot experiments, 5'-DII was measured in
isolated adipocytes as reported previously (24), and typically the
addition of 10-6 M NE to the incubation medium
induced a 3- to 4-fold increase in enzyme activity. The effect of NE
was maximal at 8 h and remained so throughout the incubation period
(data not shown).
In all experiments, each time point represents the average of the results obtained in experiments involving sets of 3–4 tubes, carried out in triplicate, for a total of 9–12 tubes per time point. Each of these triplicate tubes was handled identically until the end of incubation. Then, one tube was used to measure 3H2O incorporation into lipids, a second tube was used for measurement of ME activity, and the third tube was used for measurement of ACC activity. At the end of the incubation period, a 500-µl aliquot of cell suspension was collected from all tubes for DNA measurement as described by Giles (26). Values ranged from 8–14 µg DNA/tube and were used to calculate the lipogenesis rate and both enzyme activities. The medium from each tube was then aspirated, and the cells were processed for lipid extraction or enzymatic activities.
Hormones and drug additions
For drug addition, the substances were dissolved in volumes of
medium ranging from 10–100 µl. NE was added every 24 h along
with ascorbic acid (1 mM final) to prevent oxidation.
Appropriate control tubes containing no NE also received ascorbic acid.
T3 and T4 in the free acid
form were dissolved in 40 mM NaOH and diluted in medium to
the desired concentration. Our previous work indicated that in the
presence of 4% BSA, the dialyzable fractions of
T4 and T3 were only 1.3- to
1.4-fold the dialyzable fractions of T4 and
T3 in rat plasma (25). Iopanoic acid was
dissolved in 40% propylene glycol and then diluted in incubation
medium to a final concentration of 100 µM. Appropriate
controls had the same final concentration of propylene glycol in the
tube (0.7%).
Measurement of fatty acids synthesis by
3H2O incorporation
Rates of in vitro lipogenesis were measured in
isolated brown adipocytes as the incorporation of
3H2O into lipid by a combination of the methods
described by Saggerson and Greenbaum (30), Saggerson et al.
(31) and Folch et al. (32). Briefly,
3H2O (1.4 Ci/mol; New England Nuclear, Boston,
MA) was diluted to 50 µl with incubation medium and added to each
tube to a final specific activity of 5 µCi/ml. One hour later, cells
were extracted with chloroform-methanol (2:1; 1 ml cell suspension-10
ml of the mixture) and mixed thoroughly with 0.2 vol of a salt solution
containing 4.8 mM CaCl2, 3.6 mM
MgCl2, and 10 mM NaCl. The lower phase
containing the lipids was then transferred to a clean tube, evaporated
to dryness at 50 C, and saponified with 2 ml 5% ethanolic KOH at
70–80 C for 2 h. After 2 ml water were added to each tube, and
the ethanol was evaporated completely at 45 C, the contents of each
tube were washed with 7 ml petroleum ether followed by titration with
0.04% green bromocresol and acidification with 6 N
H2SO4 during vortexing. The lipids were then
extracted three times with petroleum ether. The combined fractions were
evaporated to dryness, redissolved in scintillation liquid (Atomlight),
and counted for 3H. The rates of lipogenesis are expressed
as microgram-atoms of 3H incorporated into lipid per h/100
µg brown adipocyte DNA.
Pilot experiments were carried out to determine the time course of 3H2O incorporation into lipids with freshly dispersed cells and with cells that had been incubated for as long as 48 h. Under both conditions the incorporation of 3H2O into lipids was fairly linear (two or three experiments per time point), indicating that the whole process was reliable, and the cells were viable up to 48 h from the dispersion time point. The basal rate of lipogenesis in cells incubated with medium alone ranged from 0.6–1.0 µg-atoms/h·100 µg DNA.
Measurement of enzyme activities
At the end of the incubations and after the medium had been
carefully aspirated, cells were resuspended in 1 ml ice-cold
homogenization buffer according to the enzyme to be assayed. For ME, we
used 10 mM Tris-HCl buffer, pH 7.4, containing 0.32
M sucrose, 2 mM EDTA, and 5 mM
2-mercaptoethanol. For ACC, we used 50 mM Tris-HCl buffer,
pH 7.5, containing 20 mM sodium citrate, 0.5 mM
EDTA, and 5 mM 2-mercaptoethanol. From this step on, all
procedures were carried out at 4 C. The cells were homogenized in a
motor-driven Teflon-glass homogenizer and centrifuged for 1 h at
100,000 x g to obtain the cytosolic fraction. All
samples were stored at -70 C for further processing.
ME was assayed by the method of Ochoa, adapted by Hsu and Lardy (33),
in 0.4 M triethanolamine buffer, pH 7.4, containing 0.03
M malate, 0.12 M MnCl2, 3.4
mM NADP+, and about 100 µg diluted cytosolic
protein, enough to cause an absorbance change of 0.05–0.1 OD/min. The
formation of the NADPH was followed for up to 5 min at 340 nm and then
plotted against time. The basal activity of ME in cells incubated with
medium alone ranged between 1.0–1.5 
OD/min·100 µg DNA.
ACC was assayed by the method of Inove and Lowenstein (34). To separate the enzyme-enriched fraction from internal cellular inhibitors, the whole citosolic fraction was size-fractionated through a 2.5 x 30-cm Sephadex G-25 column equilibrated with 1 mM dithiothreitol (DTT) dissolved in 20 mM Tris-HCl, pH 7.5. The fraction containing the enzyme was collected and diluted to 1 ml with 50 mM Tris-HCl, pH 7.5, containing 20 mM sodium citrate, 20 mM MgCl2, 1 mM DTT, and 0.5 mg/ml BSA. Subsequently, the enzyme was activated at 37 C for 30 min and immediately assayed for activity. The assay was carried out in 400 µl of a 100 mM Tris-HCl, pH 7.5, buffered solution containing 1 mM DTT, 0.2 mM acetyl-CoA, 20 mM NaH14CO3 (New England Nuclear; 42.6 Ci/mol), 5 mM ATP, 20 mM sodium citrate, 20 mM MgCl2, 0.5 mg/ml BSA, and usually 100 µl activated enzyme at 37 C for 5–10 min. The reaction was interrupted by the addition of 100 µl 4 N HCl, dried under a N2 stream, redissolved in 1 ml H2O, and then mixed with 10 ml Atomlight before counting. The results are expressed as units per 100 µg DNA; 1 U ACC activity is equal to 1 µmol [14C]malonyl CoA formed in 1 min at 37 C. The basal activity of ACC in cells incubated with medium alone ranged from 500–800 mU/100 µg DNA.
Statistical analysis
Data were analyzed by one-way ANOVA followed by multiple
comparisons using the Newman-Keuls test. Results are expressed as the
mean ± SD. For simplicity, statistical significance
is only mentioned when it is relevant to an argument.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. When no thyroid hormones were present,
the lipogenesis rate decreased progressively over time. At 12 h,
it was already 20% lower (P < 0.05), and by the
end of the incubation period, the drop had reached 45% (Fig. 1A).
ME activity also showed a tendency to decrease over time, although the
fall did not reach statistical significance (Fig. 1B). Nevertheless,
the ACC activity did decrease substantially under unstimulated
conditions; it had fallen by 30% at 12 h (P <
0.05) of incubation and by 40% at the end of the incubation period
(Fig. 1C). In the same experiment, when cells were incubated with a
near-physiological T3 concentration (0.77 x
10-9 M), the lipogenesis rate and the activity
of both enzymes (ME and ACC) continued to decrease over the incubation
period, although to a much lesser extent and not reaching statistical
significance (Fig. 1, A–C). On the other hand, when
T3 was added to the medium at a
receptor-saturating concentration, the lipogenesis rate and the
activity of both lipogenic enzymes increased markedly (Fig. 1, A–C).
By 12 h of incubation, the increment in lipogenic activity had
already reached levels 20–50% above basal values (P
< 0.05 for both ME and ACC). At the end of the incubation, the
2-fold stimulation mark had been exceeded (Fig. 1, A–C), indicating
that even during a 48-h incubation, cells retained the capacity to
respond to thyroid hormones. Interestingly enough, cells incubated in
the presence of T4 showed a shallow, but
progressive, increase in the lipogenesis rate (P <
0.05 by 36 h) and in the activity of the two lipogenic enzymes,
which tended to plateau when values were 20–35% above basal. This is
an indication that in the presence of T4, the
impact of thyroid hormones on these cells is greater than when they are
incubated with a near-physiological T3
concentration. It is therefore likely that even under these
unstimulated conditions, the basal 5'-DII activity generates enough
T3 to sustain a higher lipogenesis rate in brown
adipocytes, provided enough T4 is present. It is
worth noting that here and also in the following experiments, DNA
concentration did not decrease significantly during incubation (data
not shown), indicating that these findings represent a real change in
lipogenesis-related activity.
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shows the time course of
the lipogenesis rate and the activity of lipogenic enzymes when NE was
added to the incubation medium at a fairly high concentration
(10-6 M). The lipogenesis rate decreased
significantly by 12 h of incubation, down to 50% of the basal
values (P < 0.05), regardless of the presence of
thyroid hormones in the medium (Fig. 2A). With regard to ME activity
(Fig. 2B), the addition of NE alone resulted in only a mild decrease in
enzymatic activity (10%) by 12 h; however, the inhibition
progressed and reached 35% (P < 0.05) by 48 h of
incubation (Fig. 2B). ACC activity decreased markedly at the early time
point; it was reduced to 40% by 12 h (P <
0.05) and remained at that level throughout the incubation period (Fig. 2C). The addition of T3 at a near-physiological
concentration (0.77 x 10-9 M) did not
change the NE-mediated down-regulation of lipogenic activity during the
entire 48-h incubation period. However, as shown in Fig. 2, A–C, when
T3 was added at a receptor-saturating
concentration (154 x 10-9 M), the
NE-mediated inhibition of lipogenic activity was reversed, reaching
levels significantly higher than basal at 12 h for ME activity (up
by 25%; P < 0.05) and at 36 h for lipogenesis
rate (up by 24%; P < 0.05) and ACC activity (up by
69%; P < 0.05). In addition, when
T4 and NE were added to the incubation medium,
the stimulatory phase of lipogenic activity was more pronounced than
that observed with receptor-saturating T3
concentrations and caused a 2.2- to 2.5-fold stimulation over basal
values (Fig. 2, A–C).
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, we next
investigated the dose-response curve for NE inhibition (12-h
incubation) or stimulation (48-h incubation) of the lipogenesis rate
and for the activity of both lipogenic enzymes. Brown adipocytes were
incubated with different NE concentrations
(10-11–10-5 M) with or without
T3 or T4. Figure 3A shows
that when brown adipocytes were incubated with a wide range of NE
concentrations for only 12 h, the lipogenesis rate decreased
progressively with increasing NE concentrations in the medium.
Statistical significance was reached with 10-9
M NE when the lipogenesis rate was decreased by 35%. The
presence of a near-physiological T3 concentration
did not change this pattern, whereas T4 or high
T3 concentrations slightly shifted the curve to
the right, but were still unable to prevent the NE-mediated inhibition
of lipogenesis. Interestingly, as shown in Fig. 3B, ME activity
decreased only mildly (by about -15% at 10-5
M NE; P < 0.05) when no thyroid hormone
was added or when T3 was added at a
near-physiological concentration. Again, when either
T4 or a receptor-saturating concentration of
T3 was added, the dose-response curve was
slightly shifted to the right (Fig. 3B), minimizing the inhibitory
effect of NE on ME activity. The behavior of ACC activity was similar
to that observed for the lipogenesis rate (Fig. 3C). Regardless of the
presence of any given thyroid hormone, ACC activity decreased markedly
and progressively as the NE concentration increased. Statistical
significance was reached at 10-9 M NE, and at
10-5 M NE, ACC activity was reduced to only
30% of the basal values.
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shows
that, as expected, in the absence of thyroid hormones or in the
presence of T3 at a near-physiological
concentration, increasing NE concentrations resulted in decreased
lipogenesis and a 40–50% reduction of ME and ACC activities. On the
other hand, when brown adipocytes were incubated with a
receptor-saturating T3 concentration for 48
h, lipogenesis and both enzyme activities were stimulated up to
210%. Increasing NE concentrations tended to slightly reduce all
three lipogenic parameters, particularly ACC activity (Fig. 4C).
Nevertheless, at any given NE concentration, lipogenesis and ME and ACC
activities were well above basal values, at least by a factor of 1.7
(Fig. 4). When cells were incubated with T4, the
profiles obtained for all three parameters were startling. NE
stimulated lipogenesis and the activity of both lipogenic enzymes in a
dose-dependent fashion, exceeding 2.3-fold stimulation at the
maximum NE concentration (10-5 M).
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. As
before, in the absence of T4, NE failed to
stimulate either the lipogenesis rate or enzyme activities. On the
other hand, as anticipated from the experiments described above, the
addition of T4 to the incubation medium plus NE
resulted in stimulation of lipogenesis and both enzyme activities, and
the mechanism involved was fully blocked by the addition of IOP to the
incubation medium (Fig. 5A). A similar extent of stimulation was
detected when the cells were incubated with NE and a
receptor-saturating T3 concentration, except that
in this case IOP treatment did not blunt the lipogenic response to
T3, nor did it affect the cells when added in
combination with NE.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3–4% of
the thermogenic effects of these hormones on obligatory thermogenesis
could be accounted for by their stimulatory role in lipogenesis (3, 35).
However, less is known about the contribution of lipogenesis to
facultative thermogenesis. BAT lipogenesis accounts for a substantial
fraction of the whole rat lipogenesis (2), raising the possibility that
in this tissue, thyroid hormone-induced lipogenesis may play a more
important thermogenic role than previously recognized. Based on
published values for BAT lipogenesis (2) and oxygen consumption
of animals acclimated at room temperature (9), it has been estimated
that the caloric cost of BAT lipogenesis is 10% of the total BAT
oxygen consumption, a figure that is expected to rise during cold
stimulation.
In previous publications, we (10, 12) and others (11) have investigated in vivo the stimulation of BAT lipogenesis by the SNS and/or thyroid hormones. The results indicated, however, that these are not independent stimuli; BAT T3 production, via local T4 5'deiodination, is stimulated several-fold by the SNS (4, 36), increasing the BAT T3 concentration and its nuclear T3 receptor occupancy (5). Therefore, it was difficult to quantitate the individual role and contribution of each hormonal input because thyroid- and SNS-dependent signals are concurrently generated whenever BAT is physiologically stimulated. Another complicating factor is that thyroid hormones increase BAT cAMP production in response to NE by altering the expression of adrenergic receptors, adenylyl cyclase subtype, and other proteins involved in cAMP production (37), therefore modulating BAT sensitivity to the SNS.
The first aspect that is noteworthy about lipogenesis in isolated brown
adipocytes is that in the absence of hormonal additions to the
incubation medium, the rate of lipogenesis and the activity of ME and
ACC tended to decrease substantially over time, down to 60% by
48 h. Because cells remained viable throughout the incubation
period, these results indicate that the high rates of lipogenesis
sustained in vivo are directly dependent on neural/endocrine
stimulation. Indeed, the addition of either T4 or
T3 to the incubation medium at physiological
concentrations did minimize the time-dependent down-regulation of
lipogenesis. Furthermore, lipogenesis was stimulated up to 2-fold
when a receptor-saturating concentration of T3
was added. This confirms our in vivo data showing that
thyroid hormones positively regulate lipogenesis in the BAT (10, 12)
and indicates that T3 vigorously stimulates
lipogenesis in isolated brown adipocytes in vitro,
independently of NE.
The results obtained in the present investigation indicate that NE
modulates lipogenesis in brown adipocytes in a complex manner, and the
final biological effect depends on 1) whether T4
is present and 2) the incubation span. Alone, NE rapidly inhibited
lipogenesis in a dose-dependent fashion, whereas in the presence of a
physiological T4 concentration and longer
incubation times (48 h), NE stimulated lipogenesis 2-fold, also in a
dose-dependent manner, to a level similar to that observed when cells
were incubated with a receptor-saturating concentration of
T3. These results resemble previous findings
showing that fatty acid synthesis (38) and ACC messenger RNA levels
studied by primer extension analysis (39) decrease sharply during the
first 4–6 h of cold exposure, only to increase by a factor of 2–3
after 24–48 h of continued cold stimulation.
On the one hand, the present results confirm the capacity of NE to acutely inhibit lipogenesis, in consonance with the inhibitory effects of NE and other inducers of the cAMP pathway, such as glucagon and forskolin, on lipogenesis and/or the activity/expression of lipogenic enzymes/proteins in the liver, lactating mammary gland (15, 16, 17), BAT (40), and brown adipocytes (18, 19). On the other hand, the results obtained also indicate the capacity of NE to induce lipogenesis in isolated brown adipocytes, provided T4 is present and the incubation is allowed to proceed for 24–48 h, in accordance with the 3- to 4-fold stimulation of lipogenesis detected in vivo during cold exposure, via SNS (2).
There is extensive in vivo (10, 12) and now in
vitro evidence that the mechanism responsible for reversing the
inhibitory role of NE on BAT lipogenesis includes the activation of
5'-DII by NE. This would, in turn, intensify the impact of
T3 in this tissue by increasing the BAT nuclear
T3 receptor occupancy. As a consequence, even in
the presence of NE, lipogenesis was vigorously stimulated, as when
cells were incubated with receptor saturating concentrations of
T3. Nevertheless, the operation of such a
mechanism requires the T3 induction of
lipogenesis to be more potent than the NE inhibitory effect. Indeed,
our data strongly support this assumption. Even at the highest
concentration, NE could not inhibit or prevent the 2-fold
lipogenesis stimulation caused by receptor-saturating concentrations of
T3.
ME and ACC gene expressions are known to be up-regulated by
T3 and down-regulated by cAMP/PKA-dependent
nuclear proteins (15, 16, 17, 41). Fatty acid synthase gene expression is
also specifically inhibited by cAMP/PKA (42). Based on the time course
of our results, it is likely that an early NE-dominant phase (negative)
would be followed by a later T3-dominant phase
(positive), up-regulating the transcription of these genes. In fact, we
did report that ACC messenger RNA levels in BAT are 2-fold increased
after 48–96 h of cold stimulation (39), confirming the
T3-induced lipogenic phase. However, in addition
to these nuclear effects, ACC is known to be very sensitive to
phosphorylation by PKA. NE and other inducers of the cAMP/PKA pathway
inhibit lipogenesis, allowing PKA to inactivate the enzyme by
phosphorylation (42). This might be the reason we found a greater
inhibition of ACC activity when cells were incubated with NE (60%)
compared with the mild 10–20% inhibition of ME activity, which is
known not to be regulated by allosteric effectors or covalent
modifications. At this time we do not understand the mechanism by which
T3 would overcome ACC allosteric inactivation by
PKA.
Nonetheless, the present findings in isolated brown adipocytes confirm the idea put forward in previous publications from several groups (43, 44) and our own (5, 6, 7) that the intracellular T3 pool at any given moment represents a composite of the relative contributions of T3 derived as such from the circulation and T3 generated locally via 5'-D. This is particularly true in the BAT, given the expression of 5'-DII and the magnitude of physiological regulation it undergoes. Because 5'-DII is stimulated several-fold by NE, the local impact of thyroid hormones increases substantially, triggering the local thermogenic effects of thyroid hormones without affecting other tissues. We have shown that this mechanism is limiting for the full expression of the UCP gene (7) and the BAT thermal response during NE infusion (45).
In the present investigation we expanded this concept to include BAT lipogenesis. In this system, 5'-DII is so critical that the stimulatory effects of NE on local lipogenesis are necessarily mediated by the activation of local T4 to T3 conversion. In addition, one more piece of evidence favoring the critical role of 5'-DII in this process was obtained when isolated brown adipocytes were incubated in the presence of IOP, a specific inhibitor of T4 deiodination. The blockade of 5'-DII with IOP did not interfere with the down-regulation of lipogenesis caused by NE. However, it did blunt the NE-induced lipogenesis in these cells, emphasizing the key role of locally generated T3 in allowing stimulation of lipogenesis by NE in BAT.
Apart from the effects discussed above, the actions of NE on BAT elicit
a powerful activation of the hormone-sensitive lipase within minutes of
SNS stimulation. Lipolysis is so intense that it greatly exceeds BAT’s
capacity to oxidize fatty acids, and BAT becomes an exporter of fatty
acids (46), a fact that might contribute to fuel shivering
thermogenesis. In the present in vitro investigation we
found that during a similar time frame, the first few hours of NE
stimulation, NE decreases lipogenesis by half, indicating that the
initial sympathetic surge to BAT shifts the balance between lipolysis
and lipogenesis, stimulating the former and inhibiting the latter.
After 24 h, however, as the biological effects of the rapidly rising
local T3 production start to take place, the
inhibitory effects of NE on lipogenesis are minimized by a greater
T3 impact on the tissue, and a new steady state
is reached, where the rate of lipogenesis approaches and eventually
equals that of lipolysis. This assumption is based on previous data
(47) indicating that the size of the intracellular brown adipocyte fat
depot, as estimated by the diameter of the lipid vacuoles and cell
area, decreases by half during the first hours of cold exposure, only
to be restored by 24 h of continued cold stimulation.
T4 and the 5'-DII are critical in this process.
By stimulating several-fold the local transformation of
T4 to T3, NE induces
lipogenesis and as a consequence sustains the high rates of electron
transfer in BAT mitochondria and the capacity to export fatty
acids.
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Acknowledgments |
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Footnotes |
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Received August 20, 1997.
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References |
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 A. C. Bianco, D. Salvatore, B. Gereben, M. J. Berry, and P. R. Larsen Biochemistry, Cellular and Molecular Biology, and Physiological Roles of the Iodothyronine Selenodeiodinases Endocr. Rev., February 1, 2002; 23(1): 38 - 89. [Abstract] [Full Text] [PDF]
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 C. Curcio, A. M. Lopes, M. O. Ribeiro, O. A. Francoso Jr., S. D. Carvalho, F. B. Lima, J. E. Bicudo, and A. C. Bianco Development of Compensatory Thermogenesis in Response to Overfeeding in Hypothyroid Rats Endocrinology, August 1, 1999; 140(8): 3438 - 3443. [Abstract] [Full Text]
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 D. Salvatore, T. Bartha, and P. R. Larsen The Guanosine Monophosphate Reductase Gene Is Conserved in Rats and Its Expression Increases Rapidly in Brown Adipose Tissue during Cold Exposure J. Biol. Chem., November 20, 1998; 273(47): 31092 - 31096. [Abstract] [Full Text] [PDF]
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