1 Department of Anesthesiology and Critical Care, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts 02114; 2 Department of Psychiatry, Wayne State University School of Medicine, Detroit, Michigan 48207; and 3 Division of Endocrinology, Gerontology and Metabolism, Stanford University Medical Center, Stanford, California 94305
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Thermal injury causes a hypermetabolic state
associated with increased levels of catabolic hormones, but the
molecular bases for the metabolic abnormalities are poorly understood.
We investigated the lipolytic responses after
3-adrenoceptor
(
3-AR) agonists and evaluated
the associated changes in
-AR and its downstream signaling molecules
in adipocytes isolated from rats with thermal injury. Maximal lipolytic
responses to a specific
3-AR
agonist, BRL-37344, were significantly attenuated at post burn days
(PBD) 3 and 7. Despite significant reduction of the cell surface
3-AR number and its mRNA at PBD
3 and 7, BRL-37344 and forskolin-stimulated cAMP levels were not
decreased. Glycerol production in response to dibutyryl cAMP, a direct
stimulant of hormone-sensitive lipase (HSL) via protein kinase A (PKA),
was significantly attenuated. Although immunoblot analysis indicated no
differences in the expression and activity of PKA or in the expression
of HSL, HSL activity showed significant reductions. Finally,
3-AR-induced insulin secretion
was indeed attenuated in vivo. These studies indicate that the
3-AR system is desensitized
after burns, both in the adipocytes and in
3-AR-induced secretion of
insulin. Furthermore, these data suggest a complex and unique mechanism
underlying the altered signaling of lipolysis at the level of HSL in
animals after burns.
adrenergic receptor; burns; hormone-sensitive lipase; insulin; protein kinase A
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE HYPERMETABOLIC STATE of burn injury is associated
with uncontrolled catabolism of proteins, fats, and carbohydrates that lasts several weeks and affects morbidity and mortality. This state is
attributed in part to persistently elevated levels of catecholamines,
which under physiological conditions induce lipolysis in fat via
-adrenergic receptors (
-ARs) (31). The key regulator of lipid
metabolism in adipose tissue is the
3-AR in rodents and also
humans. Northern blot analysis, utilizing specific cDNA probes,
revealed that adipocytes possess three types of
-ARs,
1-,
2-, and
3-AR, in a proportion of
3:1:150, respectively (3). Thus the nonspecific agonist isoproterenol
induces lipolysis mainly via
3-AR, leading to breakdown of
triglyceride (TG) to free fatty acid (FFA) and glycerol. Alternatively,
BRL-37344 and CL-316234, specific agonists of
3-AR, can more effectively
induce lipolysis (1, 4). A study on genetically engineered mice with
disruption of the
3-AR gene has
confirmed that
3-AR is the
predominant receptor to mediate agonist-induced lipolysis in adipocytes
(25).
-AR-mediated lipolysis pursues the following signaling cascade.
First, ligand-bound
3-AR
activates G proteins and induces cAMP accumulation. The increased
levels of cAMP lead to activation of protein kinase A (PKA). Finally,
cAMP-dependent PKA phosphorylates and activates hormone sensitive
lipase (HSL), which catalyzes the breakdown of TG to FFA and glycerol.
After burn injury, high blood concentrations of FFA and glycerol are
observed (31).
Desensitization of receptor-mediated intracellular signaling is the
usual outcome of the presence of persistently high concentrations of a
ligand. The adenylyl cyclase (AC)-coupled
1-AR system has served as a
model system for the study of the desensitization of G protein-coupled
receptors (17). There are currently four known mechanisms of
agonist-induced desensitization that appear to have physiological
significance: receptor sequestration/internalization,
-AR kinase
(
-ARK) phosphorylation of Ser/Thr on the COOH terminus of
-AR,
PKA-mediated phosphorylation of
2-AR on
Ser261 or
Ser262, and downregulation of the
-AR. Two key factors that govern which of these mechanisms
predominate are the concentration of agonist and the time of exposure
to agonist. Short-term exposure to low concentrations of agonist in
several different cell lines causes a PKA-mediated desensitization.
Downregulation, which does not contribute to rapid desensitization,
also occurs in response to low concentrations of agonist. In response
to high concentrations of full agonists that result in high receptor
occupancy, the
-AR is rapidly desensitized through
-ARK-mediated
phosphorylation and arrestin binding, phosphorylation by PKA, and internalization.
In 3-AR, there are no consensus
phosphorylation sites for PKA and only two sites for
-ARK (18).
Nonetheless, it has been shown that the adipocytes show physiologically
induced functional desensitization (11). Fat cells isolated from
animals that have been exposed to low temperature or to high
concentrations of
-AR agonists for a prolonged period showed a
decreased responsiveness to norepinephrine in vitro, both with respect
to the extent of maximal stimulation of oxygen consumption (heat
production) and, more importantly, with respect to the norepinephrine
EC50, which was shifted
significantly to the right. The molecular mechanism behind this
functional desensitization is currently not clarified, although a
postreceptor mechanism has been proposed.
Another approach to see the specific function of
3-AR in vivo is the
3-agonist-induced elevation of
plasma insulin and glycerol levels when injected intraperitoneally (5).
In the absence of
3-AR
activity, this increase in plasma insulin levels is not observed.
BRL-37344 has also been previously shown to increase plasma insulin
levels in mice not by direct stimulation of pancreas but by release of
a heretofore-undefined factor (32, 33). This output was also utilized
for this study.
The burn-injured rat model provides a unique opportunity to investigate
the molecular mechanism whereby critical illness or stress induces
alterations of lipolytic responses to -AR ligands. The findings
observed in this model may be clinically relevant, because it examines
the cause of a common metabolic abnormality in the acute phase of
burned patients (15, 28, 29). The present study in rats with thermal
injury, using multiple molecular pharmacological approaches, examined
the efficacy of signal transduction leading to lipolysis via
-ARs,
particularly
3-AR. We found
that the
3-AR-mediated
functional response was in fact desensitized after burn, implying that
decreased clearance of lipid and/or accelerated breakdown of TG in
response to cytokines, but not enhanced response to
-AR agonists,
plays a primary role in the sustained high concentration of FFA and
glycerol in the blood of burned subjects. Detailed pharmacological
analysis revealed that both
-AR and postreceptor changes were
present after burn injury but that the etiology of decreased lipolysis
was decreased activity of HSL.
![]() |
EXPERIMENTAL PROCEDURE |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Thermal injury model. The protocol for the studies was approved by the Institutional Animal Care Committee. The animal care facility is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, as described previously (15). Male Sprague-Dawley rats (Taconic, Germantown, NY) weighing about 150-160 g were used. Six animals were used for each group (sham-burned and burn groups). After clipping of the hair, a full-thickness third-degree burn injury was produced by immersing the back of the trunk for 15 s and the abdomen for 5 s in 80°C water under pentobarbital sodium (60-70 mg/kg ip) anesthesia. A weight- and time-matched sham burn group (controls) was treated in the same manner as the trauma group, except that they were immersed in lukewarm water. Third-degree burns destroy the sensory nerve ending, making analgesia unnecessary. Both animal groups were tested at PBD 1, 3, 7, and 14.
Isolation and adipocyte preparation. At 1, 3, 7, and 14 days after thermal or sham injury, the epididymal adipose tissues were removed under anesthesia and immediately used to prepare isolated adipocytes by use of the collagenase method (23). One gram of tissue was minced with scissors and digested in 2 ml of HEPES-Krebs-Ringer buffer A (110 mM NaCl, 10 mM KCl, 2 mM MgCl2, 25 mM NaHCO3, 25 mM HEPES/NaOH, 1.7 mM CaCl2, 5 mM glucose, 0.3% bovine serum albumin, and 100 µM ascorbic acid saturated with 95% O2-5% CO2 gas) containing 1 mg/ml of type II collagenase (Sigma) at 37°C for 45 min under constant shaking (100 cycles/min) in a 50-ml Falcon tube. After digestion, adipocytes were filtered through a double-layered nylon mesh and centrifuged at 400 g for 1 min at room temperature. The infranatant was aspirated by Pasteur pipette and washed four times with buffer B (buffer A with 1 mM CaCl2). The survivability of isolated fat cells was confirmed by the absence of trypan blue staining in over 90% of the cells. After aspiration, the cells were resuspended in 20× volume of buffer B in a 50-ml Falcon tube and gently stirred at 37°C until further experiments.
Lipolysis assay.
Lipolysis was assessed according to the method of Tebar et al. (26).
After isolation of adipocytes, plastic vials (6-ml plastic sample
vials, Wheaton, Millville, NJ) containing 100 µl of five different
concentrations of agonists, isoproterenol, BRL-37344 (provided by Dr.
M. A. Cawthorne, Smith-Klein-Beecham, UK), forskolin, or dibutyryl cAMP
(DBcAMP) were placed in a modular incubator chamber
(Billups-Rothenberg, Del Mar, CA) at 37°C. A 400-µl aliquot of
well-stirred cell suspension was added to each tube, the chamber was
closed, and the cell suspension was incubated at 37°C under constant shaking and oxygenation with
O2-CO2
(95:5) for 60 min. The vials were then placed in ice water for 5 min,
and 360 µl of infranatant were transferred to a new tube and mixed
with 40 µl of 30% perchloric acid to give a final concentration of
3%. The tubes were kept on ice for 20 min, after which they were
centrifuged at 25,000 g for 5 min at
4°C. A 350-µl amount of supernatant was transferred to a new
tube, 35 µl of 5 N KOH were added to adjust the pH to 9.5-9.8,
and the supernatant was stored at 20°C until further
experiments. Glycerol concentration was enzymatically measured using
glycerokinase and NAD-dependent glycerophosphate dehydrogenase,
according to the method of Wieland (30). The data were normalized to
protein concentration and genomic DNA content. The protein
concentration was determined by the Bradford method and that of genomic
DNA by Hoechst 33258 dye.
[125I]iodo-()-cyanopindolol
binding assay.
The binding assay was performed according to Feve et al. (6) with minor
modification. The adipose tissues were extracted from the animals,
frozen immediately in liquid nitrogen, and stored at
80°C.
On the day of the experiment, the tissues were homogenized in hypotonic
buffer [10 mM HEPES-NaOH (pH 7.4), 1 mM EDTA, 20 µg/ml
aprotinin, 50 µg/ml leupeptin, 0.5 µg/ml pepstatin, and 1 mM
phenylmethylsulfonyl fluoride (PMSF)] with a Polytron homogenizer (model PT 10/35, Brinkmann Instruments) operated at maximum speed for
30 s and were kept on ice for 5 min. The fat cake was removed by
centrifugation at 15,000 g for 15 min
at 4°C. The pellet was resuspended in 1 ml of the same buffer, and
the nuclear fraction was separated by centrifugation at 1,000 g for 10 min. The
supernatant-containing membrane was transferred to a new
microcentrifuge tube and centrifuged at 17,000 g for 20 min at 4°C. The pellet
was again resuspended in 1 ml of the same buffer and centrifuged at
17,000 g for 20 min. The pellet was
resuspended in 500 µl of the same buffer, and its protein
concentration was determined by the Bradford method and was used as the
purified membrane fraction. A 20-µg sample of the membrane was used
for the saturation binding experiments on
-AR, with
[125I]iodo-(
)-cyanopindolol
(ICYP) 2,200 Ci/mmol, Dupont-NEN as the specific ligand for
-AR. A
concentration range of 1-4,000 pM of ICYP, with or without 100 µM propranolol (Sigma) in binding buffer [20 mM
Tris · HCl (pH 7.5), 0.1% BSA, 5 mM glucose, 1 mM MgCl2, and 1 mM PMSF] at
37°C for 20 min was used. After centrifugation at 17,000 g for 10 min, the pelleted membranes
were washed once with 1 ml of the same binding buffer. After
centrifugation at 17,000 g for 10 min,
the pellet was cut from the tube, and the radioactivity bound to
membrane was counted. Specific binding was defined as total binding
minus nonspecific binding. The equilibrium dissociation constants
(KD) and the
maximal number of binding sites
(Bmax) were calculated with
EBNA-LIGAND programs (19).
RIA for cAMP production.
A 500-µl aliquot of stirred cell suspension was incubated with 10 µM isoproterenol or 50 µM forskolin at 37°C for 5 min in the
presence of 1 µg/ml adenosine deaminase. The reaction was terminated
by the addition of 50 µl of 100% trichloroacetic acid to give a
final concentration of 5% and by immediate placement of the vials on
ice. The samples were frozen at 80°C until further experiments. At the time of the next experiment, samples were centrifuged at 15,000 rpm for 10 min at 4°C to collect supernatant. The cAMP levels in the supernatant were measured using a cAMP assay kit
(Biotrak, Amersham). The cAMP levels were normalized by the total DNA
amount in each sample, and the values were shown as nanograms of cAMP
per milligram DNA per minute.
Northern blotting analysis of -ARs.
Cytosolic RNA from the adipose tissues, previously frozen, was
extracted using guanidium thiocyanate solution, followed by centrifugation in cesium chloride solutions. Total RNA (20 µg) was
applied on a 1.2% agarose gel (SeaKem). The electrophoresed agarose
gel was partially denatured in 0.1 N NaOH and transferred to nylon
transfer membrane (MSI) by the microcapillary method by use of
20× standard sodium citrate (SSC). The membrane was baked and
flashed with ultraviolet light. The cDNA probes specific to rat
1- or
3-AR mRNA (9, 10) were labeled
with [
-32P]dCTP
(3,000 Ci/mmol, Du Pont-NEN) by Klenow fragment with a random primer
(Prime It-II, Stratagene) and were purified using a DNA purification
column (Bio-Rad). The membrane was hybridized with each probe at
42°C for 12-24 h, washed once with 0.5% SDS-2× SSC for
10 min at 42°C, and then washed three times with 0.5% SDS-0.1× SSC for 30 min at 42°C. The expression of
1- or
3-AR mRNA was visualized by
autoradiography and quantified using PhosphoImager (model BAS 2000, Fuji, Japan). All of the data were normalized by
-actin mRNA
expression with a murine
-actin cDNA probe.
Western blotting of PKA C-subunit and HSL. A 50-µg aliquot of homogenate from adipose tissue was subjected to SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Bio-Rad). After the membrane had been blocked with 2% skim milk, 2% BSA, and 0.01% sodium azide in PBS for 1 h at room temperature, the membrane was incubated with anti-PKA C-subunit rabbit polyclonal serum (Upstate Biotechnology) at 1 µg/ml for overnight at 4°C or anti-HSL rabbit polyclonal serum (16) at 1:10,000 dilution for 1 h at room temperature. The membrane was washed with PBS containing 0.05% Tween 20 (PBST) and incubated with anti-rabbit IgG goat polyclonal conjugated peroxidase (Bio-Rad) at 1:10,000 dilution in PBST for 90 min at room temperature. The membrane was washed five times with PBST, and antigenic bands were visualized by chemiluminescence (Amersham).
Determination of PKA activity. PKA activity was measured according to Goueli et al. (8). One gram of adipose tissue was homogenized using precooled Polytron homogenizer in 5 ml of homogenizing buffer (50 mM Tris · HCl, pH 7.5, 5 mM EDTA, 1 mM PMSF, 2 µg/ml aprotinin, 10 µM leupeptin, and 0.5 µg/ml pepstatin A). The homogenates were centrifuged at 40,000 g for 10 min at 4°C, and the supernatant was kept on ice before an assay for kinase activity. PKA activity was measured by the PKA Assay System (Promega) according to the manufacturer's recommended procedure. Five micromoles of cAMP were used as agonist for stimulation.
Determination of HSL activity. HSL activity was measured according to Kraemer et al. (16). One gram of adipose tissue was washed three times with ice-cold PBS and homogenized using a precooled Polytron homogenizer in 5 ml of 0.25 M sucrose, 1 mM EDTA, 2 µg/ml aprotinin, 1 mM PMSF, and 50 mM Tris · HCl (pH 7.0). After the homogenate was centrifuged at 40,000 g for 45 min, the infranatant was passed through glass wool and the protein concentration was determined. Aliquots of 50 µg were assayed in triplicate for neutral cholesteryl esterase activity with cholesteryl-[1-14C]oleate (Du Pont-NEN).
Measurement of plasma insulin after BRL-37344 injection. On PBD 3, rats were fasted for 14-18 h and anesthetized by injection of pentobarbital (50 µg/ml ip) and kept on a heating pad to keep warm for 20 min. BRL-37344 (1 mg/kg ip) in 300 µl of saline was carefully injected, and whole blood was collected from the heart after 15 min with a heparinized and EDTA-treated syringe. Plasma insulin level was measured using the Rat Insulin Kit (Linco).
Statistics. All experiments were performed in at least three to six independent experiments. Student's t-test was used to test significance.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The effect of thermal injury on lipolytic responses in adipocytes.
To characterize the effect of burn injury on lipid kinetics, we first
measured 3-AR agonist-induced
glycerol production in adipocytes prepared from rats with thermal
injury at PBD 1, 3, 7, and 14. Figure 1
demonstrates the dose-dependent effects of the specific
3-AR agonist BRL-37344 on
glycerol production in sham or burned animals. In the control group,
maximal stimulation (a 5-fold increment over the basal level) was
achieved at 1 µM BRL-37344 with an
EC50 value of 1-4.5 nM,
consistent with the reported literature (5). At PBD 1, no significant
change in BRL-37344-stimulated glycerol production was observed between burned and sham-burned rats. At PBD 3 and 7, BRL-37344-induced glycerol
production was significantly attenuated compared with sham-burn rats.
Maximal glycerol production induced by 1 µM BRL-37344 was reduced 75 and 67% in burned rats compared with sham-burned rats at PBD 3 and 7, respectively. At PBD 3, the EC50
value for glycerol production was also significantly increased 3.5-fold in burned rats compared with sham-burned rats. At PBD 14, maximal glycerol production was similar in the burn and sham groups.
|
Effect of thermal injury on binding kinetics of
-ARs.
Next, we examined the
-AR number in the adipose tissues to see
whether this attenuation of lipolysis after burn injury was due to
downregulation of receptor number.
[125I]ICYP was used as
the ligand, and the binding properties of
3-AR were calculated from the
low-affinity binding sites. At PBD 3, the period when inhibition of
3-AR agonist-induced glycerol
production was reduced, the Bmax
for
3-AR was significantly
reduced to 46% compared with that of sham rats (Table
1). The
KD value was not altered. The Bmax of
3-AR was still decreased to
35% at PBD 7. At PBD 14 the Bmax
was still reduced, but the
KD value was also significantly reduced to 50% of that of sham rats. The reduced KD, therefore,
could potentially facilitate coupling of
3-AR and G proteins and is
consistent with the finding that glycerol production was normalized at
PBD 14. Thus it was tentatively concluded that alteration of receptor
kinetics could explain the attenuated lipolysis or desensitized
responses to
3-AR agonists.
|
1-AR and
3-AR mRNA levels.
The changes in ICYP kinetics observed after burns prompted us to
examine whether this downregulation occurred at the transcriptional level of
-AR. The
1-AR and
3-AR mRNA expression in adipose tissue was assessed by Northern blotting by use of specific cDNA probes
for rat
1-AR and
3-AR (9). At PBD 1,
3-AR mRNA levels in burned rats
were significantly increased relative to sham-burned rats (Fig.
2). The levels of these transcripts
normalized at PBD 3 and were significantly decreased at PBD 7 and 14. These latter changes in transcripts thus paralleled the decrease in
3-AR number at PBD 7 and 14. No
difference in
1-AR mRNA level
was observed in burn rats at PBD 1. At PBD 3, the
1-AR mRNA level was
significantly decreased in burned rats. At PBD 7 and 14, however, the
1-AR mRNA levels in burned rats
were significantly increased, which may be a compensatory response to
decreased
3-AR expression, as
documented previously by Susulic et al. (25). Although these decreases
in
1-AR and
3-AR mRNA well explain the
decreased number of these receptors as determined by ICYP binding, we
cannot rule out the possibility of decreased mRNA stability after
burns. The mechanism of decreased
1-AR and
3-AR mRNA level may be due to either transcriptional regulation or destabilization of the mRNA.
|
Thermal injury and cAMP production.
The alteration of receptor binding kinetics and mRNA level of
3-AR prompted us to examine the
effect of isoproterenol on cAMP production in adipocytes at PBD 3 and
7. To characterize
-AR function via and beyond the receptor,
isoproterenol and forskolin-mediated cAMP production was examined in
isolated adipocytes. On both days, forskolin-induced cAMP production
was not significantly changed (Fig. 3),
suggesting that adenylyl cyclase was intact in adipocytes after burn.
It is noteworthy that even a slight increase of cAMP production is
sufficient to cause full activation of HSL (13). For lipolysis to be
reduced to 20% at PBD 3, as observed in our study, a drastic reduction
of cAMP production by isoproterenol would be necessary. As illustrated
in Fig. 3, however, isoproterenol-induced cAMP production in burn rats
was reduced to only 50% of that of sham rats, and that too was
observed only at PBD 3. This reduction would not be sufficient to cause
80% reduction of lipolysis. It can thus be concluded that receptor
number reduction would not be the reason for the desensitization of
3-AR-mediated impairment in
lipolysis after burn. This notion was further supported by the
surprising finding that at PBD 7, isoproterenol-induced cAMP production
was significantly augmented, twofold compared with that of sham rats.
Because isoproterenol-induced glycerol production was significantly
attenuated at PBD 7, the possibility that receptor number reduction or
altered AC activity as the cause for impairment of
3-AR signaling was totally
excluded.
|
Analysis of downstream signaling molecules of lipolysis.
Next, the effect of DBcAMP, which stimulates PKA directly to cause
lipolysis, in bypassing the upstream signaling molecules (i.e.,
3-AR and AC), was examined. As
expected, DBcAMP-induced glycerol production of burn rats was
significantly attenuated by 70 and 60% compared with sham rats at both
PBD 3 and 7 (Fig. 4). This impairment was
therefore comparable to that observed during the
stimulation with isoproterenol or BRL. Thus this
experiment confirms that the inhibition of adrenoceptor agonist-induced
lipolysis was not caused by abnormalities in signaling molecules
between
3-AR and AC and points
to aberrations further downstream, such as the effector molecules, PKA
and/or HSL.
|
Analysis of PKA and HSL expression and their activity.
To assess the efficacy of a further downstream signaling pathway, we
quantitated the expression of the catalytic subunit of PKA, PKA-C, and
HSL in adipose tissue by Western blotting. At PBD 3, there were no
significant differences between sham-burned and burned rats in
expression of PKA-C, which migrated at 40 kDa (Fig.
5B).
Similarly, no differences were observed between groups in the
expression of HSL, which migrated at 84 kDa (Fig.
5A). Thus one can conclude that the
decreased lipolysis was not due to downregulation or decreased
expression of these downstream molecules. Next, the PKA activity of
homogenates prepared from adipose tissues was tested (Fig.
6). There was no significant difference in
basal PKA activity between sham (360 pmol · mg1 · min
1)
and burned rats (400 pmol · mg
1 · min
1)
at PBD 3. Exogenous cAMP clearly stimulated PKA activity equally (3.5-fold) in both groups. Thus differences in PKA activity between the
groups cannot explain the differences in lipolysis. In additional experiments, the HSL activity using cholesteryl
[1-14C]oleate, which
is a specific substrate for HSL, was tested (Fig. 7). The basal HSL activity was 150 nmol · mg
1 · h
1
in sham rats and was significantly suppressed to 98 nmol · mg
1 · h
1
in burned rats at PBD 3 (P < 0.02).
These data demonstrate that the decreased adipocyte lipolysis after
burn injury was mainly due to depressed HSL activation and not to the
decreased expression of PKA or HSL or to decreased activation of PKA.
|
|
|
Effect of BRL-37344 on plasma insulin level, in vivo.
In the following study we examined whether the decreased function of
3-AR is also observed when the
rats are stimulated with BRL, in vivo. CL-316,243, another
3-AR agonist, specifically increased plasma insulin and glycerol levels when injected
intraperitoneally (5). In the absence of
3-AR activity, this increase in
plasma insulin levels is not observed. BRL has also been previously
shown to increase plasma insulin levels in mice, not by direct
stimulation of the pancreas but by release of a heretofore-undefined
factor (32, 33). We therefore injected 1 mg/kg of BRL intraperitoneally and extracted a venous blood sample 15 min after injection at PBD 3. The plasma insulin level was 0.24 ng/ml in sham rats and increased
20-fold to 4.6 ng/ml after BRL injection (Fig.
8). In burned rats, the basal plasma
insulin level was the same as that of sham rats but increased only
fivefold (1.3 ng/ml), which was only 30% of the response seen in sham
rats (P < 0.005). These data clearly
demonstrated that
3-AR-mediated
signaling, indirectly assayed as release of insulin into plasma, was
also desensitized in vivo.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In normal humans, lipids constitute 80% of the stored energy.
Consequently, when an individual must rely on endogenous fuel supplies
for energy, such as those occurring in the acute phase of burn injury
when enteral nutrition is difficult, lipids are physiologically the
most desirable source of energy. Thus the optimal stress response would
involve mobilization and use of fat, so that lean body mass is spared.
The current study in rats has established that the important signaling
pathway for lipolysis, the 3-AR
system, is desensitized after burn injury at PBD 3 and 7 and reverts to
normal by PBD 14. In other words, mobilization of fat, important for
optimal stress response, is impaired at certain periods after burn
injury. Additional salient findings of this study are that
1)
3-AR number is decreased at PBD
3, 7 and 14; 2) AC and PKA activity
is unimpaired; 3) lipolysis induced by DBcAMP, which directly activates PKA and HSL, was also impaired similarly to that observed for receptor-mediated lipolysis; and, most
importantly, 4) HSL activity was
also decreased in burned animals.
These in vitro findings may be viewed as contradictory to the in vivo
data of Wolfe et al. (31). With use of stable isotopes, their study in
humans documented increased rate of appearance of glycerol in burned
patients compared with normal subjects. It is important to note,
however, that in the study by Wolfe et al., the demographics of the
patient population were quite diverse: 1) the burned patients consisted of
10 children and 8 adults; 2) the
body surface burn ranged from 40 to 95%;
3) the patients studied were between
9 and 48 (mean 20 ± 4.5) days after burn; and
4) the response to exogenous
epinephrine was also quite variable, desensitized or normal. It is
important to note, however, that both the study by Wolfe et al. and our
own study do not suggest that -AR-mediated lipolytic responses are
completely absent. In our study in rats, resensitization occurred by
PBD 14. The precise period in which resensitization occurs in humans is
difficult to predict, as this could be influenced by many clinical
factors, including age, burn size, and other complicating factors. On
the basis of in vivo human studies, it is evident that some patients are desensitized whereas others are resensitized (31). It is important
to know, however, if the
-AR is desensitized or resensitized, as
this has relevance and implications for the use of
-AR agonists and
antagonists in the treatment of burn injury.
One might, therefore, pose the question as to why measured plasma FFA
and glycerol levels are high in burn patients and rodents in those
periods when 3-AR signaling is
desensitized. Glucagon also stimulates lipolysis via the same cAMP
pathway in adipose tissues, and serum glucagon levels are increased
after burns. Because our study shows that desensitization is presumably
located at the PKA-HSL level, it is less likely that glucagon is
responsible for enhanced lipolysis after burns. One possibility is that
the hepatic clearance of lipolysis breakdown products is impaired, because liver is the primary organ where lipid clearance occurs. In
fact, liver dysfunction is a common problem after burns, as evidenced
by biochemical, clinical, metabolic, or drug clearance indexes. The
other possibility is that mechanism(s) other than the
-AR-mediated
lipolysis occur in adipose tissue, particularly in stress states.
Indeed, previous studies have demonstrated that some cytokines induce a
coordinate catabolic response in adipose cells that leads to decreased
fat storage or inhibition of lipolysis (4, 12). Tumor necrosis factor
(TNF), interleukin-1 (IL-1), interferon-
(IFN-
), and IFN-
decrease lipoprotein lipase activity and increase lipolysis in
adipocytes. The molecular mechanisms whereby these cytokines stimulate
lipolysis or impair lipogenesis are not fully understood. TNF-
or
IL-1 in vitro results in complete loss of stimulatory effect of insulin
on glucose transport and a dose-dependent stimulation of lipolysis,
assessed by glycerol release by up to 400% above controls (4).
Furthermore, these lipid changes were obliterated by the administration
of anti-TNF antibody or the use of mice genetically not susceptible to
lipopolysaccharide (28). It is well documented that levels of
cytokines, including TNF, are increased in burned patients and rodents
after burn injury (20). Thus the enhanced lipolysis, assessed by
glycerol turnover documented in vivo by others, may essentially be
related to nonadrenergic-mediated lipolysis.
There is evidence for desensitized
3-AR response in the presence
of high circulating concentrations of
-AR agonists (18). In one
study, rats were continuously infused with isoproterenol (50 or 100 µg · kg
1 · h
1)
for 3 days by osmotic minipumps, and epididymal adipocytes were isolated and analyzed for
-AR-mediated lipolysis (18). Cells from
isoproterenol-treated animals were desensitized to the lipolytic effect. Binding of
[125I]ICYP was
decreased by ~80% in adipocyte plasma membranes isolated from
treated rats, indicating that
-ARs were downregulated. Cellular concentrations of G proteins were not altered.
Our study can be compared and contrasted to the above findings. We
observed that 1) the -AR number
on the adipocyte surface was significantly reduced. We did not measure
catecholamines in the circulation or locally during our experiments,
but others have documented that persistent elevations of catecholamines
do occur in subjects with burn injury (31). The persistent elevations of catecholamines seen in burned subjects may have played a role in the
downregulation of
-AR. These findings are therefore consistent with
those previously reported from our laboratory, of desensitization of
the myocardial
-AR system after burn injury to the rat (29). In this
regard, our present findings on the rat adipocyte are therefore similar
to those observed with isoproterenol infusion and its effect on
-AR-induced lipolysis (18). In our study, the
-AR-mediated cAMP
production was intact, as was the PKA activity in adipocytes. The
DBcAMP-induced lipolysis was, however, attenuated, as was the HSL
activity. In this regard it has been observed that adipocytes from
spontaneously hypertensive rats demonstrated a blunted lipolytic
response to both isoproterenol and DBcAMP, suggestive of a similar
defect in regulation of lipolytic enzymes at the PKA-HSL level (21). A
similar defect of decreased lipolysis due to a defect at the HSL levels
was indirectly documented in familial combined hyperlipidemia (22).
These two studies, however, did not characterize changes in PKA/HSL
activity or expression, but the changes, similar to those observed
after burns, may have been present in the spontaneously hypertensive
rats and in familial hyperlipidemia.
The disparate relationship between unaltered AC-mediated cAMP
production and the blunted cAMP-induced lipolysis needs explanation. Previous studies have reported BRL, compared with isoproterenol, to be
a potent agonist for lipolysis, but only as partial agonist for cAMP
production (13, 14, 24). This discrepancy between cAMP production and
lipolysis has been observed for many years, suggesting an alternative
pathway for lipolysis to that via cAMP (14, 24). It is speculated that
this mechanism involves HSL but not AC, whereby -AR stimulation
induces HSL activation via a non-PKA pathway (13). Whether this
alternative pathway was activated was not investigated. These
experiments together, however, lead to the conclusion that the major
defect in the lipolytic machinery is at the level of HSL.
The other possible mechanism is the poor compartmentalization of cAMP, PKA, and HSL in adipocytes. It is known that A kinase anchor proteins (AKAPs) immobilize and concentrate PKA at specific intracellular locations in other tissues. So far, the role of AKAPs in adipocytes is hardly studied but should be focused on in the future.
The reduction of -AR number does not necessarily mean that signaling
via this receptor is decreased. In certain receptor signaling systems,
just 10% of the total cell surface receptors is sufficient for the
full capacity of signal transduction (18). It therefore is not
surprising to observe the discrepancy between the reduced
-AR number
and normal receptor-mediated cAMP production. It is an interesting
possibility, however, that the activity of the discussed positive
regulator might be tightly correlated with the receptor availability,
explaining the reduced activity of HSL in burn. Another important point
that should be mentioned is that activity of HSL is regulated by its
state of phosphorylation. HSL is phosphorylated not only at
Ser563 by PKA but also at
Ser565 by other kinases. These
kinases include glycogen synthase kinase-4, Ca2+/calmodulin-dependent kinase
II, and AMP-dependent protein kinase. Phosphorylation of
Ser565 prevents the
phosphorylation of Ser563 in vitro
(7). It is possible, therefore, that TNF or other cytokines associated
with burn injury increase the basal phosphorylation of HSL at
Ser565 and thus prevent its
activation by PKA. Our burn model may provide a unique opportunity to
investigate the in vivo importance of phosphorylation of HSL at
Ser565 and its contribution to the
desensitization of PKA-dependent activation of HSL.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. M. A. Cawthorne for providing BRL-37344 and Dr. K. Yonezawa (Kobe University) for setting up the lipolysis assay.
![]() |
FOOTNOTES |
---|
This work was supported in part by National Institutes of Health (NIH) Grants GM-55081-3 and GM-31569-17 to J. A. J. Martyn, MH-56036 to T. Okamoto, and from The Department of Veterans Affairs and NIH Grant DK-46942 to F. B. Kraemer.
Present address for T. Ikezu and T. Okamoto: Dept. of Neurosciences, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave. NC30, Cleveland, OH 44195.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for correspondence and reprint requests: J. A. J. Martyn, Dept. of Anesthesia, Massachusetts General Hospital, Boston, MA 02114.
Received 26 January 1999; accepted in final form 8 April 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Arch, J. R.,
A. T. Ainsworth,
M. A. Cawthorne,
B. Piercy,
M. W. Sennitt,
V. E. Thody,
C. Wilson,
and
S. Wilson.
Atypical beta-adrenoceptor on brown adipocytes as target for antiobesity drugs.
Nature
309:
163-165,
1984[Medline].
2.
Bloom, J. D.,
M. D. Dutia,
B. D. Johnson,
A. Wissner,
M. C. Burns,
E. E. Largis,
J. A. Dolan,
and
T. H. Claus.
Disodium (R,R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino] propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL 316,243). A potent beta-adrenergic agonist vertually specific for beta 3 receptors. A promising antidiabetic and antiobesity agent.
J. Med. Chem.
35:
3081-3084,
1992[Medline].
3.
Collins, S.,
K. W. Daniel,
E. M. Rohlfs,
V. Ramkmar,
I. L. Taylor,
and
T. W. Gettys.
Impaired expression and functional activity of the beta 3- and beta 1 adrenergic receptors in adipose tissue of congenitally obese (C57BL/6J ob/ob) mice.
Mol. Endocrinol.
8:
518-527,
1994[Abstract].
4.
Doerrler, W.,
K. R. Feingold,
and
C. Grunfeld.
Cytokines induce catabolic effects in cultured adipocytes by multiple mechanism.
Cytokine
6:
478-484,
1994[Medline].
5.
Emorine, L. J.,
S. Marullo,
M.-M. Briend-Sutren,
G. Patey,
K. Tate,
C. Delavier-Klutchko,
and
A. D. Strosberg.
Molecular characterization of the human beta 3-adrenergic receptor.
Science
245:
1118-1121,
1989[Medline].
6.
Feve, B.,
L. J. Emorine,
F. Lasnier,
N. Blin,
B. Baude,
C. Nahmias,
A. D. Stroberg,
and
J. Pailault.
Atypical beta-adrenergic receptor in 3T3-F442A adipocytes. Pharmacological and molecular relationship with the human beta 3-adrenergic receptor.
J. Biol. Chem.
266:
20329-20336,
1991
7.
Garton, A. J.,
and
S. J. Yeaman.
Identification and role of the basal phosphorylation site on hormone sensitive lipase.
Eur. J. Biochem.
191:
245-250,
1990[Abstract].
8.
Goueli, B. S.,
K. Hisao,
A. Tereba,
and
S. A. Goueli.
A novel and simple method to assay the activity of individual protein kinases in a crude tissue extract.
Anal. Biochem.
225:
10-17,
1995[Medline].
9.
Granneman, J. G.,
and
K. N. Lahners.
Differential adrenergic regulation of beta 1- and beta 3-adrenoceptor messenger ribonucleic acids in adipose tissues.
Endocrinology
130:
109-114,
1992[Abstract].
10.
Granneman, J. G.,
K. N. Larners,
and
A. Chaudhry.
Molecular cloning and expression of the rat beta 3-adrenergic receptor.
Mol. Pharmacol.
40:
895-899,
1991[Abstract].
11.
Green, A.,
R. M. Carroll,
and
S. B. Dobias.
Desensitization of -adrenergic receptors in adipocytes causes increased insulin sensitivity of glucose transport.
Am. J. Physiol.
271 (Endocrinol. Metab. 34):
E271-E276,
1996
12.
Hauner, H.,
T. Petruschke,
M. Russ,
K. Rohrig,
and
J. Eckel.
Effects of tumour necrosis factor alpha (TNF alpha) on glucose transport and lipid metabolism of newly differeniated human fat cells in cell culture.
Diabetologia
38:
764-771,
1995[Medline].
13.
Hollenga, C.,
F. Brouwer,
and
J. Zaagsma.
Differences in functional cyclic AMP compartments mediating lipolysis by isoprenaline and BRL 37344 in four adipocyte types.
Br. J. Pharmacol.
102:
577-580,
1991[Abstract].
14.
Hollenga, C.,
F. Brouwer,
and
J. Zaagsma.
Differences in functional cyclic AMP compartments mediating lipolysis by isoprenaline and BRL 37344 in four adipocyte types.
Eur. J. Pharmacol.
200:
325-330,
1991[Medline].
15.
Ikezu, T.,
T. Okamoto,
K. Yonezawa,
R. G. Tompkins,
and
J. A. J. Martyn.
Analysis of thermal injury-induced insulin resistance in rodents. Implication of postreceptor mechanisms.
J. Biol. Chem.
272:
25289-25295,
1997
16.
Kraemer, F. B.,
S. Patel,
M. S. Saedi,
and
C. Sztalryd.
Detection of hormone-sensitive lipase in various tissues. I. Expression of an HSL/bacterial fusion protein and generation of anti-HSL antibodies.
J. Lipid Res.
34:
663-671,
1993[Abstract].
17.
Lefkowitz, R. J.,
J. Pitcher,
K. Krueger,
and
Y. Daaka.
Mechanisms of beta-adrenergic receptor desensitization and resensitization.
Adv. Pharmacol.
42:
416-420,
1998[Medline].
18.
Liggett, S. B.,
N. J. Freedman,
D. A. Schwinn,
and
R. J. Lefkowitz.
Structural basis for receptor subtype-specific regulation revealed by a chimeric beta 3/beta 2-adrenergic receptor.
Proc. Natl. Acad. Sci. USA
90:
3665-3669,
1993[Abstract].
19.
MacPherson, G. A.
Analysis of radioligand binding experiments. A collection of computer programs for the IBM PC.
J. Pharmacol. Methods
14:
213-228,
1985[Medline].
20.
Monafo, W. W.
Initial management of burns.
N. Engl. J. Med.
335:
1581-1586,
1996
21.
Nelson, K. M.,
R. E. Shepherd,
and
J. A. Spitzer.
Lipolysis and beta-adrenergic receptor binding on adipocytes of spontaneously hypertensive rats.
Biochem. Med. Metab. Biol.
37:
51-60,
1987[Medline].
22.
Reynisdotter, S.,
M. Eriksson,
B. Anelin,
and
P. Arner.
Impaired activation of adipocyte lipolysis in familial combined hyperlipidemia.
J. Clin. Invest.
95:
2161-2169,
1995[Medline].
23.
Rodbell, M.
Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis.
J. Biol. Chem.
239:
375-380,
1964
24.
Rohlfs, E. M.,
K. W. Daniel,
R. T. Premont,
L. P. Kozak,
and
S. Collins.
Regulation of the uncoupling protein gene (Ucp) by beta 1, beta 2, and beta 3-adrenergic receptor subtypes in immortalized brown adipose cell lines.
J. Biol. Chem.
270:
10723-10732,
1995
25.
Susulic, V. S.,
R. C. Frederich,
J. Lawitts,
E. Tozzo,
B. B. Kahn,
M. Harper,
J. Himms-Hagen,
J. S. Flier,
and
B. B. Lowell.
Targeted disruption of the beta 3-adrenergic receptor gene.
J. Biol. Chem.
270:
29483-29492,
1995
26.
Tebar, F.,
I. Ramirez,
and
M. Soley.
Epidermal growth factor modulates the lipolytic action of catecholamines in rat adipocyte. Involvement of a Gi protein.
J. Biol. Chem.
268:
17199-17204,
1993
27.
Van Liefde, I.,
A. Van Witzenburg,
and
G. Vuquelin.
Multiple beta adrenergic receptor subclasses mediate the I-isoproterenol-induced lipolytic response in rat adipocytes.
J. Pharmacol. Exp. Ther.
262:
552-558,
1992[Abstract].
28.
Vega, G. L.,
and
C. R. Baxter.
Tumor necrosis factor mediates hypertriglyceridemia during thermal injury in mice genetically susceptible to lipopolysaccharides.
J. Burn Care Rehab.
12:
463-467,
1991[Medline].
29.
Wang, C.,
and
J. A. J. Martyn.
Burn injury alters beta adrenergic receptor and second messenger function in rat ventricular muscle.
Crit. Care Med.
24:
118-124,
1996[Medline].
30.
Wieland, O. H.
Glycerol: UV-method.
In: Methods in Enzymatic Analysis, edited by H. U. Bergmeyer. New York: Academic, 1984, vol. 3, p. 504-510.
31.
Wolfe, R. R.,
D. N. Herndon,
F. Jahoor,
H. Miyoshi,
and
M. Wolfe.
Effect of severe burn injury on substrate cycling by glucose and fatty acids.
New Engl. J. Med.
317:
403-408,
1987[Abstract].
32.
Yoshida, T.
The antidiabetic beta 3-adrenoceptor agonist BRL 26830A works by release of endogenous insulin.
Am. J. Clin. Nutr.
55:
237S-241S,
1992[Abstract].
33.
Yoshida, T.,
N. Hiraoka,
and
M. Kondo.
Effects of a beta 3-adrenoceptor agonist, BRL 26830A, on insulin and glucagon release in mice.
Endocrinol. Jpn.
38:
641-646,
1991[Medline].
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |