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Department of Pharmacology, Chemotherapy, and Medical Toxicology, LITA Ospedale L. Sacco, Milan University, School of Medicine, Milan, Italy
Address all correspondence and requests for reprints to: Dr. Enzo Nisoli, Dipartimento di Farmacologia, Chemioterapia e Tossicologia Medica, Università degli Studi, Via Vanvitelli 32, 20129 Milan, Italy.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The thermogenic activity of BAT, evoked by norepinephrine (NE) secreted from the sympathetic nerves that innervate richly and directly the brown adipocytes as well as BAT blood vessels (for review, see 4 requires a very high perfusion rate through its vascular system for the supply of oxygen and substrate to the mitochondria, the respiration rate of which is extremely high, and for export of the heat produced. In contrast to what occurs in many other organs, an increase in sympathetic activity in brown fat induces vasodilation (5). Heat, the major metabolic product of brown fat seems unlikely to be of major significance as a modulator of the tissue’s blood flow, as at constant heat production, simply changing the concentration of oxygen in arterial blood changes the blood flow (6). In addition, the increase in blood flow that accompanies the NE-induced thermogenesis in BAT is considerably independent of the concentration of NE to which the tissue’s vascular smooth muscle is exposed (6, 7). Accordingly, it has been proposed that brown fat vasodilation may be controlled by a vasoactive substance (a vasodilator) produced by the adipocytes (7, 8).
Nitric oxide (NO) is a ubiquitous paracrine messenger for blood vessels. It accounts for the activity of endothelium-derived relaxing factor, which stimulates vasodilation by releasing NO from the endothelium, which, in turn, acts on adjacent smooth muscle (9). NO originates not only from endothelial cells, but also from adventitial nerves and epithelial cells, where it mediates endothelium-independent smooth muscle relaxation (10, 11). In addition, neurons use NO to match cerebral blood flow with neuronal activity; similarly, bronchial epithelial and endothelial cells use NO to match ventilation and perfusion (12). Thus, it was of interest to investigate whether brown adipocytes produce NO, which, in turn, mediates NE-induced vasodilation in BAT, to match thermogenesis and perfusion.
To address this question, we investigated the presence and activity of NO synthase (NOS) in BAT and brown fat cells of the rat. We conclude that inducible NOS (iNOS) is expressed in and NO is released from brown adipocytes, that their production appears to be under the stimulatory control of NE, and that this noradrenergic control is active in physiological conditions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adipose cell culture and treatment
Three million cells were added to 24-well culture plates
(Nunclon Delta). The cells were cultured in 2.0 ml of a culture medium
consisting of DMEM supplemented with 4 mM glutamine, 10%
newborn calf serum, 4 nM insulin, and 10 mM
HEPES, with 50 IU penicillin, 50 µg streptomycin, and 25 µg sodium
ascorbate/ml (all from Flow Laboratories, Milan, Italy), at 37 C in a
water-saturated atmosphere of 6% CO2 in air. The medium
was completely exchanged with fresh prewarmed medium on day 1 (when the
cultures were first washed with 5 ml prewarmed DMEM) and on days 3 and
9 (without wash). In experiments for determination of NOS activity, the
cells were exposed to NE (freshly diluted in buffers containing 0.1%
ascorbic acid) for 24 h and then harvested. In experiments
studying the modulation of iNOS synthesis, cells were exposed to NE for
4–6 h.
Nitrite analysis
The appearance of NOS activity was monitored by the increase in
the concentration of nitrite (NO2-), a
breakdown product of NO, in the cell culture medium after treatment
with the various drugs. After the indicated incubation time, a 100-µl
aliquot of the culture medium was mixed with 50 µl Griess reagent
(1% sulfanilamide, 0.1% naphthylenediamine dihydrochloride, and 2.5%
H3PO4) and incubated at room temperature for 10
min, and the optical density was determined in a microplate reader at
570 nm. Specific activity is given as nanomoles of
NO2- produced per mg protein. Fresh culture
medium served as the blank, and solutions of
NaNO2- diluted in culture medium were used as
standards.
Assay of brown adipocyte iNOS activity
Rat brown adipocyte iNOS activity was estimated by measuring the
conversion of L-2,3,4,5-[14C]arginine to
L-2,3-[14C]citrulline according to the
modification of the method described by Bredt et al. (16).
Brown adipocytes (1 x 108), untreated or treated with
1 µM NE for 24 h, were homogenized with an
Ultra-Turrax homogenizer (5-mm blade) for 20 sec in 0.5 ml of a buffer
containing 50 mM HEPES (pH 7.4), 1 mM
DL-dithiothreitol, 1 mM EDTA, 0.5 µg/ml
leupeptin, 2 µg/ml aprotinin, 0.7 µg/ml pepstatin, and 1
mM phenylmethylsulfonylfluoride. After centrifugation
(12,000 x g, 20 min at 4 C), an aliquot of the
supernatant was added to a reaction mixture at a final volume of 100
µl containing 50 mM HEPES (pH 7.4), 50 nCi
L-[14C]arginine (310 mCi/mmol; Amersham,
Milan, Italy), 1 µM arginine, 1 mM NADPH, 1
mM EDTA, 1 mM EGTA, 10 µM FAD,
0.1 mM (6R)-5,6,7,8-tetrahydrobiopterin. The
mixture was incubated for various periods of time at 37 C. The reaction
was stopped by adding 0.4 ml of a 1:1 slurry of Dowex AG 50W-X8 (Fluka,
Milan, Italy; Na+ form) in 50 mM HEPES, pH 5.5,
and, after 15 min of shaking, radioactivity in the supernatant was
measured. Enzyme activity was expressed as nanomoles of citrulline
formed by 1 mg protein.
PCR assay
Total cytoplasmic RNA was isolated from 1 x
106 cultured cells using the RNAzol method (TM Cinna
Scientific, Friendswood, TX). For PCR analysis, RNAs were treated for
1 h at 37 C with 6 U ribonuclease (RNase)-free deoxyribonuclease
I/µg RNA in 100 mM Tris-HCl, pH 7.5, and 50
mM MgCl2 in the presence of 2 U/µl placental
RNase inhibitor. The concentration of RNA was determined by absorbance
at 260 nm, and the integrity of the RNA was confirmed by
electrophoresis through 1% agarose gels containing 0.1 µg/ml
ethidium bromide. One microgram of total RNAs was converted to
complementary DNA (cDNA) with 200 U Moloney murine leukemia virus
reverse transcriptase (Promega, Madison, WI) in 20 µl of the
buffer supplied by Promega buffer containing 0.4 mM
deoxy-NTP, 2 U/ml RNase inhibitor, and 0.8 µg
oligo(deoxythymidine)15 primer (Promega). A control without
reverse transcriptase was performed for each sample to verify that
amplification did not proceed from residual genomic DNA. An aliquot
(5%) of the resulting cDNA was amplified using the reverse primer
((5'-CCACAATAGTACAATACTACTTGG-3'; calculated Tm = 66 C) and forward
primer (5'-ACGAGGTGTTCAGCGTGCTCCACG-3'; calculated Tm = 78 C) to yield
a 395-bp PCR product (17), with truncated Thermus acquaticus
DNA polymerase (Biotaq, Bioprobe Systems, Milan, Italy) in 50 µl
standard buffer [20 mM Tris-HCl (pH 8.55), 16
mM (NH4)2SO4, 2.5
mM MgCl2, 150 µg/ml BSA, and 200
µM deoxy-NTP]. PCR conditions were as follows:
denaturation at 94 C for 30 sec, annealing at 60 C for 45 sec, and
polymerization at 72 C for 45 sec. After 35 cycles, a final 10-min
incubation at 72 C was carried out. After amplification, 18 µl of the
reaction mixture were separated by electrophoresis (2.0% agarose gel
in Tris-borate-EDTA buffer containing 0.1 µg/ml ethidium bromide).
The identity of the PCR products was confirmed by hybridization using
an internal oligonucleotide (data not shown). The messenger RNA (mRNA)
for the constitutive ß-actin was examined as the reference cellular
transcript. ß-Actin mRNA amplification products were present at
equivalent levels in all cell lysates. The reaction was performed using
specific primers as described previously (18).
Western blotting
For Western blotting, 100 µg immunoprecipitated protein
extracts were resolved by 8% SDS-PAGE and transferred to
nitrocellulose filter papers. The primary antibody (monoclonal antibody
directed against the mouse macrophage iNOS at a dilution of 1:250;
Affiniti Research Products, Mamhead, UK) was diluted in Tris-buffered
saline, pH 7.6, and incubated with membranes overnight at 4 C.
Immunoreactive bands were visualized by enhanced chemiluminescence
according to the specifications of the manufacturer (Amersham). For
quantitation, densitometric measurements were performed with the LKB
UltroScan XL laser densitometer (LKB, Rockville, MD).
Immunoprecipitation
Cells were scraped in lysis buffer containing 50 mM
Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1%
Nonidet P-40, 0.5% sodium deoxycholate, 1 mM
phenylmethylsulfonylfluoride, 5 µg/ml leupeptin, 0.7 µg/ml
pepstatin, and 1 µg/ml aprotinin. The suspension was sonicated for 30
sec at full power and centrifuged at 12,000 x g for 10
min at 4 C. The resulting supernatant was isolated, the protein content
was determined by the BCA protein assay (Pierce Chemical Co., Rockford,
IL), and the pellet was discarded. Adipose tissue samples (100 mg) were
cut into small pieces with a razor blade and resuspended in 3 vol lysis
buffer. Samples were then sonicated and centrifuged as described above.
The resulting lipids floating to the top of the sample were discarded
as were the pellets. The aqueous infranatant was retained, and protein
content was determined as described above. Monoclonal antibody to mouse
macrophage iNOS (2 µg) was added to 1 ml (500 µg) brown fat cell or
BAT solubilized samples. After a 60-min incubation at 4 C, 50 µl
protein G-Sepharose (Boehringer Mannheim, Mannheim, Germany) were added
to precipitate antibody. Protein G pellets were washed three times with
buffers in accordance with the manufacturer’s recommended protocol.
Immunoprecipitated proteins were denatured with loading buffer,
consisting of 2-fold concentrated Laemmli SDS sample buffer (50
mM Tris-HCl pH 6.8; 100 mM dithiothreitol; 2%
SDS; 0.1% bromophenol blue; and 10% glycerol), in a boiling water
bath for 3 min and resolved by SDS-PAGE.
Data analysis
Data are reported as the mean ± SEM of n
independent determinations. All experiments were performed at least
three times, each time with three or more independent observations.
Comparisons were made using one-way ANOVA followed by
Student-Newman-Keuls post-hoc comparisons, and
P < 0.01 vs. the control value was
considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Nitrite was
first detected between 4–6 h of continuous exposure to NE and
continued to increase linearly up to 48 h, the longer period
tested. Accumulation was 11- and 25-fold greater than basal levels at
24 and 48 h, respectively. The response to NE was dose dependent,
with 50% of maximal nitrite accumulation occurring at a NE dose of
about 25 nM (when measured after 24 h of incubation;
Fig. 1B). Maximal accumulation occurs at a NE concentration of 100
µM. These results indicate that nitrite accumulation in
the culture medium, most likely as a consequence of NOS activity (see
below), can be induced by NE in confluent brown fat cells.
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This NOS activity was only slightly reduced when the assay was carried
out in the presence of 1 mM EGTA and the absence of any
added Ca2+ or calmodulin (90 ± 3% vs. no
EGTA; n = 3). Cytoplasmic extracts from control cells (no
stimulus) did not exhibit NOS activity. These results indicate that
induction of nitrite production in brown fat cells is a consequence of
a Ca2+/calmodulin-independent NOS activity comparable to
the inducible form of the enzyme characteristic of activated
macrophages.
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Figure 3A shows that iNOS mRNA levels were concentration
dependently increased by NE treatment (4 h), and that this effect was
already evident at 0.01 nM NE. As a control of the NE
effect on the brown fat cells, its ability to induce an intense UCP
gene expression, as previously reported (18, 19), was also verified
(data not shown).
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125
kDa that was detected in brown adipocytes exposed to 100 nM
NE (Fig. 3B
). This band was not detected in unstimulated cells. These
results contribute, for the first time to our knowledge, to show that
an iNOS with molecular and antigenic properties to the murine
macrophage iNOS is induced by NE in rat brown adipocytes. Of note, the affinities of iNOS mRNA and protein responses to NE differ considerably from each other. A possible reason for this could be simply the different sensitivities of the detection methods used to measure the two parameters (i.e. PCR is more sensitive than Western blotting). On the other hand, however, it cannot be excluded that more complex phenomena (e.g. different mRNA and protein turnover, different coupling between cAMP and mRNA or protein synthesis) are involved.
Characterization of the adrenergic receptor subtypes involved in
iNOS and NO induction
To pharmacologically characterize the adrenergic receptor
subtype(s) involved in NE action, iNOS mRNA and NO levels were
quantified in cultured brown adipocytes after treatment with specific
adrenergic compounds. Figure 4 shows that exposure of
cells for 4–6 h (mRNA) or 24 h (nitrite) to selective agonists of
ß1-adrenoceptors (dobutamine, 1 µM),
ß2-adrenoceptors (salbutamol, 1 µM), or
ß3-adrenoceptors (CGP 12177A, 10 µM)
induced iNOS mRNA and NO production. These inductions were lower than
that induced by NE (1 µM). In addition, the nonselective
ß-adrenoceptor antagonist, propranolol, at 100 µM was
able to only partially block the NE-induced iNOS mRNA and NO
production. These findings may suggest that 
-adrenoceptors are also
involved in the effects of NE. A more detailed pharmacological
characterization of the adrenoceptor subtypes involved is now being
performed.
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clearly shows the stimulatory effect of 1
µM forskolin on iNOS mRNA expression and NO production in
brown adipocytes. These findings firstly demonstrate that NE induces
iNOS expression in brown adipocytes at least in part through the
increases in cAMP induced by the stimulation of ß-adrenoceptors
present on the plasma membrane.
Effects of actinomycin D on NE induction of NOS activity
To assess whether the increased accumulation of NOS depended upon
de novo transcription, brown fat cell cultures were
incubated with NE in the presence of the transcriptional inhibitor
actinomycin D. Over 90% of the nitrite accumulation elicited by NE
exposure (1 µM; 24 h) was blocked when actinomycin D
(1 µg/ml) was present throughout the incubation period (mean ±
SEM, 31.5 ± 5.2 vs. 3.05 ± 0.3 nmol
nitrite/mg protein; n = 4). A short pulse of NE (2 h, followed by
22 h in the absence of NE) resulted in nitrite accumulation
(10.5 ± 1.8 nmol/mg protein) that was 33% of that obtained by
24 h of continuous exposure. When actinomycin was present during
the 2-h pulse, there was no subsequent detectable nitrite accumulation.
These results indicate that NE induction of NOS activity occurs at the
level of gene transcription, and that a brief exposure to NE is
sufficient to initiate NOS expression.
Regulation of iNOS in vivo
The culture studies suggested that iNOS mRNA can be dynamically
modulated in brown adipocytes by NE, implying that this is an important
mechanism by which NO may be regulated in vivo. To
investigate this, rats were exposed to a low temperature environment (4
C) for 48 h, and iNOS mRNA and protein were investigated by PCR
and immunoblotting in different BAT preparations. During cold exposure
there is an increase in the activity of the sympathetic nerves
innervating BAT. This induces hypertrophy of BAT, which enhances the
animal’s capacity for nonshivering thermogenesis (21), and is usually
accompanied by an increase in its blood flow (3, 22). The levels of
iNOS mRNA were poorly detectable in BAT of room temperature-exposed
control animals, but were much higher in BAT of animals exposed for
48 h at low temperature (Fig. 5A). Similar results
were obtained in collagenase-dispersed mature brown adipocytes of the
two groups of rats (data not shown). The appearance of iNOS in
cold-exposed rats was confirmed by Western blot analysis under reducing
conditions (Fig. 5B). In addition, the accumulation of
L-[14C]citrulline was not detectable in
cytosol of BAT obtained from rats acclimated to room temperature,
whereas exposure to low temperature induced NOS activity (0.38 ±
0.06 nmol/mg protein). These results contribute, for the first time to
our knowledge, to show that an iNOS, with molecular and antigenic
properties similar to those of the murine macrophage iNOS, is induced
in BAT by increased sympathetic activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NOSs, the enzymes responsible for synthesis of NO, are homodimers whose
monomers are themselves two enzymes fused, a cytochrome reductase and a
cytochrome that requires three cosubstrates (L-arginine,
NADPH, and O2) and five cofactors or prosthetic groups
(flavin adenine diclonucleotide, flavin mononucleotide, calmodulin,
tetrahydrobiopterin, and heme) (23). Several distinct NOS isoforms have
been described and shown to represent the products of three distinct
genes (24). These include two constitutive
Ca2+/calmodulin-dependent forms of NOS, including ncNOS
(also designated NOS1), whose activity was first identified in neurons
and maps at 12q24.2, and ecNOS (also designated NOS3), first identified
in endothelial cells and mapping at 7q35-36. The inducible form of NOS,
iNOS (also designated NOS2), is Ca2+ independent, is
expressed in a broad range of cell types, and maps to 17cen-q12 (23).
Induction of iNOS has been demonstrated in many cell types, including
macrophages, neutrophils, hepatocytes, vascular smooth muscle cells,
endothelial cells, cardiac myocytes, and islet cells (23, 24). Stimuli
for iNOS induction include, but are not limited to, bacterial
lipopolysaccharide (LPS), interferon-
, and tumor necrosis factors.
Cloning and sequencing of the 5'-region upstream of exon 1 from the
iNOS gene of the mouse and human (25, 26) revealed the presence of
numerous prototypical consensus sequences for genes that are
transcriptionally activated by cytokine, including two sites for
nuclear factor-
B (NF-B). Other stimuli that induce iNOS activity
and are assumed, but not proven, to act at the level of transcription,
include cAMP-elevating agents (27, 28). Because these agents share with
LPS the capacity to elicit nuclear translocation of NF-B directly or
via the generation of reactive oxygen radical intermediates, it is
tempting to speculate that NF-B is their common mediator. Our
findings suggest that iNOS synthesis is induced in brown fat cells by
NE at least in part via ß1-, ß2-, and
ß3-adrenoceptors, probably through increased cAMP
production. Indeed, a cAMP-elevating agent such as forskolin induced
iNOS mRNA expression in differentiated adipocytes. In resting cells,
expression of iNOS is usually absent.
There are several lines of evidence showing that the L-arginine-NO pathway plays an important role in regulating the microcirculation of various kinds of vasculature, such as the rat gastric mucosa (29), kidney (30), hamster cheek pouch (31), and human forearm (32). However, its role has not been conclusively defined in BAT. Our study demonstrates that exposure of the rat to cold environments induced iNOS mRNA production and activity in BAT. Thus, NO could be involved in vasodilation accompanying cold-induced thermogenesis of brown fat. Indeed, BAT is a highly vascularized organ, strategically localized along the vascular tree, that is involved in nonshivering thermogenesis in most species of animals. Brown fat exhibits a very wide range of blood flow rates, from 0.1–1.0 ml/g·min in the unstimulated tissue to 5–28 ml/g·min when tissue is stimulated by exogenous catecholamines (33). These increases of sometimes as much as 50-fold in the rate of brown fat blood flow clearly implies control of blood flow by changes in the tone of the smooth muscle of the tissue’s resistance vessels (small arteries and arterioles) and perhaps also precapillary sphincters. The resistance vessels of BAT are abundantly supplied by noradrenergic nerve fibers, which would suggest that the sympathetic nervous system may have some role in the control of brown fat blood flow. However, as summarized by Foster (6), the vasodilation in BAT accompanying activation of thermogenesis may be largely or entirely independent of a change in the activity of the vascular sympathetic nerves. It has been well demonstrated that the increase in blood flow that accompanies NE-induced thermogenesis in BAT is considerably independent of the concentration of NE to which the tissue’s vascular smooth muscle is exposed. Accordingly, Foster and Depocas (7) maintained that vasodilation in BAT is secondary to the NE-induced activation of thermogenesis in the adipocytes, and they suggested that brown fat blood flow may be controlled by a vasoactive substance (a vasodilator) produced by the adipocytes. Numerous hypotheses for these vasodilator substances have been proposed. For example, H+, K+, hyperosmolarity, the cytosolic redox state of the NAD+/NADH system, and the ratio of ATP/ADP have been suggested as mediators of the metabolically coupled vasodilation in brown fat (34, 35). However, no conclusive evidence exists for any of them.
That NO could be a key coupling factor that links changes in blood flow and metabolism of BAT was suggested by Nagashima et al. (36), who observed that the NOS blocker L-NAME completely abolished the NE-induced increase in blood flow through BAT and significantly inhibited the NE-increased temperature of both BAT and colon in urethan-anesthetized rats. Up until now, however, NE-induced iNOS and NO production had never been demonstrated directly, as shown here in rat brown adipocytes.
In conclusion, our results show that iNOS is synthesized in and NO is released from brown adipocytes, and their production is directly dependent on sympathetic activity. Our findings also suggest that iNOS expression can be dynamically modulated in BAT in vivo, as acute cold exposure, by increasing NE output, increased both iNOS mRNA and NOS activity in rat. This mechanism seems to match thermogenesis and the perfusion rate in BAT.
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Acknowledgments |
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Footnotes |
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Received July 19, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and ß-adrenergic induction of the
expression of the uncoupling protein thermogenin in brown adipocytes
differentiated in culture. J Biol Chem 265:16464–16471This article has been cited by other articles:
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| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
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, Basal;
•, NE-induced. In B, the basal nitrite level has been subtracted.
). Data represent the mean ±
SEM of at least three experiments.
