A unique mechanism of desensitization to lipolysis mediated by beta 3-adrenoceptor in rats with thermal injury

Tsuneya Ikezu1, Shingo Yasuhara1, James G. Granneman2, Fredric B. Kraemer3, Takashi Okamoto1, Ronald G. Tompkins1, and Jeevendra A. J. Martyn1

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURE
RESULTS
DISCUSSION
REFERENCES

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 beta 3-adrenoceptor (beta 3-AR) agonists and evaluated the associated changes in beta -AR and its downstream signaling molecules in adipocytes isolated from rats with thermal injury. Maximal lipolytic responses to a specific beta 3-AR agonist, BRL-37344, were significantly attenuated at post burn days (PBD) 3 and 7. Despite significant reduction of the cell surface beta 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, beta 3-AR-induced insulin secretion was indeed attenuated in vivo. These studies indicate that the beta 3-AR system is desensitized after burns, both in the adipocytes and in beta 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURE
RESULTS
DISCUSSION
REFERENCES

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 beta -adrenergic receptors (beta -ARs) (31). The key regulator of lipid metabolism in adipose tissue is the beta 3-AR in rodents and also humans. Northern blot analysis, utilizing specific cDNA probes, revealed that adipocytes possess three types of beta -ARs, beta 1-, beta 2-, and beta 3-AR, in a proportion of 3:1:150, respectively (3). Thus the nonspecific agonist isoproterenol induces lipolysis mainly via beta 3-AR, leading to breakdown of triglyceride (TG) to free fatty acid (FFA) and glycerol. Alternatively, BRL-37344 and CL-316234, specific agonists of beta 3-AR, can more effectively induce lipolysis (1, 4). A study on genetically engineered mice with disruption of the beta 3-AR gene has confirmed that beta 3-AR is the predominant receptor to mediate agonist-induced lipolysis in adipocytes (25).

beta -AR-mediated lipolysis pursues the following signaling cascade. First, ligand-bound beta 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 beta 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, beta -AR kinase (beta -ARK) phosphorylation of Ser/Thr on the COOH terminus of beta -AR, PKA-mediated phosphorylation of beta 2-AR on Ser261 or Ser262, and downregulation of the beta -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 beta -AR is rapidly desensitized through beta -ARK-mediated phosphorylation and arrestin binding, phosphorylation by PKA, and internalization.

In beta 3-AR, there are no consensus phosphorylation sites for PKA and only two sites for beta -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 beta -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 beta 3-AR in vivo is the beta 3-agonist-induced elevation of plasma insulin and glycerol levels when injected intraperitoneally (5). In the absence of beta 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 beta -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 beta -ARs, particularly beta 3-AR. We found that the beta 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 beta -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 beta -AR and postreceptor changes were present after burn injury but that the etiology of decreased lipolysis was decreased activity of HSL.


    EXPERIMENTAL PROCEDURE
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURE
RESULTS
DISCUSSION
REFERENCES

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 beta -AR, with [125I]iodo-(-)-cyanopindolol (ICYP) 2,200 Ci/mmol, Dupont-NEN as the specific ligand for beta -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 beta -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 beta 1- or beta 3-AR mRNA (9, 10) were labeled with [gamma -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 beta 1- or beta 3-AR mRNA was visualized by autoradiography and quantified using PhosphoImager (model BAS 2000, Fuji, Japan). All of the data were normalized by beta -actin mRNA expression with a murine beta -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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURE
RESULTS
DISCUSSION
REFERENCES

The effect of thermal injury on lipolytic responses in adipocytes. To characterize the effect of burn injury on lipid kinetics, we first measured beta 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 beta 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.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of BRL-37344 on glycerol production in isolated adipocytes. Dose-response curves of BRL-37344-stimulated glycerol production (nmol/mg protein) are presented. open circle , Control groups; , burned groups. Adipocytes were isolated from epididymal fat pads of burned animals at various periods [post burn days (PBD) 1, 3, 7, and 14] or of sham animals, and glycerol release was measured. Three animals were examined in each group, and 3 independent experiments were performed in each animal at each period. Values are representative data obtained from 1 animal expressed as means ± SE of 3 samples. beta 3-adrenoceptor (beta 3-AR)-mediated lipolysis was impaired at PBD 3 and 7 and reverted to normal by PBD 14. * Significant differences in glycerol release between sham and burned animals for the same concentration of agonist (P < 0.05).

Isoproterenol, a nonspecific beta -AR agonist, showed a response similar to that of BRL-37344 in both time and magnitude of attenuation, although the EC50 values for glycerol production were not significantly changed (data not shown). These findings thus indicate that, after burn injury, isoproterenol and BRL-37344-induced lipolysis was impaired in a subacute manner. This impaired lipolytic response could be due to altered beta 3-AR or to postreceptor signaling.

Effect of thermal injury on binding kinetics of beta -ARs. Next, we examined the beta -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 beta 3-AR were calculated from the low-affinity binding sites. At PBD 3, the period when inhibition of beta 3-AR agonist-induced glycerol production was reduced, the Bmax for beta 3-AR was significantly reduced to 46% compared with that of sham rats (Table 1). The KD value was not altered. The Bmax of beta 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 beta 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 beta 3-AR agonists.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   ICYP binding kinetics of beta 1-AR and beta 3-AR

As mice disrupted of the beta 3-AR gene show significant increase of beta 1-AR mRNA (5), we next examined whether beta 1-AR binding kinetics were also altered in burned rats because of the burn-induced downregulation of beta 3-AR. The binding properties of beta 1-AR were obtained as the high-affinity sites of [125I]ICYP binding. As shown in Table 1, the Bmax values of beta 1-AR were reduced rather than increased and were significantly reduced to 40% of that of sham rats at PBD 7.

beta 1-AR and beta 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 beta -AR. The beta 1-AR and beta 3-AR mRNA expression in adipose tissue was assessed by Northern blotting by use of specific cDNA probes for rat beta 1-AR and beta 3-AR (9). At PBD 1, beta 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 beta 3-AR number at PBD 7 and 14. No difference in beta 1-AR mRNA level was observed in burn rats at PBD 1. At PBD 3, the beta 1-AR mRNA level was significantly decreased in burned rats. At PBD 7 and 14, however, the beta 1-AR mRNA levels in burned rats were significantly increased, which may be a compensatory response to decreased beta 3-AR expression, as documented previously by Susulic et al. (25). Although these decreases in beta 1-AR and beta 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 beta 1-AR and beta 3-AR mRNA level may be due to either transcriptional regulation or destabilization of the mRNA.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 2.   beta 1-AR and beta 3-AR mRNA levels in rat adipose tissues after burn injury. Open bars, control groups; closed bars, burned groups. mRNA levels were determined by Northern blotting with 20 µg of cytosolic RNA isolated from epididymal adipose tissue of burned or sham-burned rats at 1, 3, 7, and 14 PBD. Six rats were examined in each group at each period. Blots of burned rats were quantified by PhosphoImager, normalized by beta -actin mRNA expression, and then expressed as a fraction (%mean ± SE) of those from corresponding values of sham rats. ND, not significantly different. *, **, and *** Significant differences between burned and time-matched sham-burned rats (P < 0.05, 0.01, and 0.02, respectively).

Thermal injury and cAMP production. The alteration of receptor binding kinetics and mRNA level of beta 3-AR prompted us to examine the effect of isoproterenol on cAMP production in adipocytes at PBD 3 and 7. To characterize beta -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 beta 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 beta 3-AR signaling was totally excluded.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3.   cAMP production from adipocytes in response to isoproterenol (ISO, A) and forskolin (FSK, B). Open bars, control groups; closed bars, burned groups. Isolated adipocytes were obtained from rat epididymal fat pads. cAMP production was measured in burned and sham rats in the presence of ISO (10 µM) or FSK (50 µM) at 3 and 7 PBD. The amount of cAMP obtained was normalized by DNA concentration of each sample. Values are means ± SE for 6 rats/group expressed as a percentage of cAMP production relative to sham rats; 3 independent experiments were performed in each group at each period. * Significant differences between sham and burn groups (P < 0.05) at the same time points. Values corresponding to 100% stand for 3.27 ± 0.46 (3 PBD, ISO), 0.88 ± 0.29 (7 PBD, ISO), 9.84 ± 0.95 (3 PBD, FSK), and 3.99 ± 0.96 (7 PBD, FSK) ng · mg DNA-1 · min-1. ND, not significantly different. These results suggest that cAMP production was unimpaired after burns.

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., beta 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 beta 3-AR and AC and points to aberrations further downstream, such as the effector molecules, PKA and/or HSL.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of dibutyryl cAMP (DBcAMP) on lipolysis in adipocytes. Open bars, control groups; closed bars, burned groups. Glycerol production of isolated adipocytes was measured in burned and sham-burned rats at PBD 1, 3, and 7 in the presence of 1 mM DBcAMP. Six rats were examined, and 3 independent experiments were performed in each group at each period. Results (means ± SE) were normalized to protein concentration in each sample and are shown as a percentage of those seen in corresponding sham rats. ** or *** Significant differences between sham and burn groups (P < 0.02 and P < 0.01, respectively) at the same time point. Values corresponding to 100% stand for 81.5 ± 19.5 (1 PBD), 81.3 ± 28.8 (3 PBD), and 109.6 ± 9.31 (7 PBD) nmol · mg protein-1 · 60 min-1. Thus DBcAMP-induced lipolysis was also impaired at PBD 3 and 7, similar to that of beta 3-AR-mediated responses.

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 · mg-1 · 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.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 5.   Expression of protein kinase A subunit C (PKA-C) and hormone-sensitive lipase (HSL) in adipose tissues. The amount of PKA-C and HSL in adipose tissues was measured by Western blotting. Six burned and 6 sham-burned rats were tested at PBD 3 for expression of these proteins. A: HSL migrated at 84 kDa (kD) in 10% gel. B: PKA-C migrated at 40 kDa in 4-20% gradient gel. Lane C is positive control (0.1 ng of bovine PKA-C, Calbiochem). No differences in protein expression of PKA or HSL were observed between sham and burn groups.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 6.   PKA activity in adipose tissues. Open bars, control groups; closed bars, burned groups. PKA activity of homogenate of adipose tissue was measured in 6 burned and 6 sham-burned rats at PBD 3 in the presence of 5 mM cAMP. Results were expressed as pmol · mg-1 · min-1. No differences (ND) in basal or stimulated PKA activity were observed. 32P-PO4, phosphorylation assessed by incorporation of radioactivity.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 7.   HSL activity in adipose tissues. Open bar, control groups; closed bar, burned groups. HSL activity of homogenate of adipose tissue was measured in 6 burned and 6 sham-burned rats at PBD 3. Results were expressed as nmol of substrate hydrolyzed · mg-1 · h-1. HSL activity was significantly decreased in adipose tissue from burned animals. ** Significant differences between sham and burn groups (P < 0.02).

Effect of BRL-37344 on plasma insulin level, in vivo. In the following study we examined whether the decreased function of beta 3-AR is also observed when the rats are stimulated with BRL, in vivo. CL-316,243, another beta 3-AR agonist, specifically increased plasma insulin and glycerol levels when injected intraperitoneally (5). In the absence of beta 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 beta 3-AR-mediated signaling, indirectly assayed as release of insulin into plasma, was also desensitized in vivo.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 8.   Plasma insulin level after BRL-37344 injection in vivo. Open bars, control groups; closed bars, burned groups. Plasma insulin levels were measured in 6 burned and 6 sham-burned rats at PBD 3 before and after intraperitoneal injection of BRL. Results were shown as ng/ml of serum insulin levels. Each column shows a different group of rats. Release of insulin by BRL was inhibited in burned animals at PBD 3, suggestive of defective beta 3-AR signaling in vivo.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURE
RESULTS
DISCUSSION
REFERENCES

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 beta 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) beta 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 beta -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 beta -AR is desensitized or resensitized, as this has relevance and implications for the use of beta -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 beta 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 beta -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-alpha (IFN-alpha ), and IFN-gamma 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-alpha 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 beta 3-AR response in the presence of high circulating concentrations of beta -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 beta -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 beta -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 beta -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 beta -AR. These findings are therefore consistent with those previously reported from our laboratory, of desensitization of the myocardial beta -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 beta -AR-induced lipolysis (18). In our study, the beta -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 beta -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 beta -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 beta -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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURE
RESULTS
DISCUSSION
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[Abstract/Free Full Text].

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 beta -adrenergic receptors in adipocytes causes increased insulin sensitivity of glucose transport. Am. J. Physiol. 271 (Endocrinol. Metab. 34): E271-E276, 1996[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Free Full Text].

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[Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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].


Am J Physiol Endocrinol Metab 277(2):E316-E324
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Ikezu, T.
Articles by Martyn, J. A. J.
Articles citing this Article
PubMed
PubMed Citation
Articles by Ikezu, T.
Articles by Martyn, J. A. J.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online