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Experimental Studies |
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000
Address all correspondence and requests for reprints to: Cynthia M. Arbeeny, Ph.D., Sepracor Pharmaceuticals, 111 Locke Drive, Marlborough, Massachusetts 01752. E-mail: carbeeny{at}sepracor.com
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BRL35135, a potent ß3-AR agonist in rodents, has generated controversial data concerning the role of ß3-AR in mediating a lipolytic response in human white adipocytes (12, 13). Ligget (14) has shown differences in rat and human ß3-ARs using BRL37344 (the active, deesterified metabolite of BRL35135). BRL37344 was a full agonist in Chinese hamster ovary (CHO) cells transfected with rat ß3-AR; however, it was a partial agonist in CHO cells transfected with human ß3-AR. Clinical studies with healthy volunteers has indicated that the lipolytic response elicited by BRL35135 was mediated by the ß2-AR (15), in contrast to studies in rodents, in which this response in mediated by the ß3-AR (2, 3). Therefore, the lack of activity of BRL37344 in human adipocytes may relate to its poor efficacy for the human ß3-AR, rather than lack of ß3-AR expression in human adipocytes. Results from recent studies have further suggested that the ß3-AR plays a role in human adipocyte function (16, 17). A defect in the ß3-AR may contribute to the obese diabetic phenotype in Pima Indians and other populations (16). Furthermore, overexpression of ß3-AR activity, as assessed by the stimulation of lipolysis by CGP12177, has been found in omental adipocytes from obese individuals (17).
Most studies of ß-AR activity in humans has been limited to WAT; however, little is known concerning the functional role of BAT in adult humans. BAT functions in rodents and newborn humans to generate body heat (18). This is achieved by the activity of UCP, which uncouples the oxidation of fatty acids from phosphorylation of ATP. The presence of BAT or the reactivation of BAT in adult humans has been reported by several researchers (19, 20, 21). However, quantitation of BAT in humans has been difficult, because brown adipocytes (identified by the presence of UCP) exist within depots of white adipocytes. In vivo measurements of thermogenic activity have also indicated that BAT is present in humans (22).
The purpose of the present study was to establish a nonhuman primate model to study BAT metabolism. The cynomolgus monkey was chosen because its lipolytic response to various pharmacological agents in white adipocytes is similar to that in humans (8). The role of each ß-AR subtype in mediating lipolysis and oxygen consumption in this tissue was determined, and a comparison with lipolysis in white adipocytes was made. The results of this study contribute to the understanding of BAT function in a nonhuman primate and suggest that this tissue may play a role in whole body energy balance in primates.
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Materials and Methods |
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The axillary brown adipose and abdominal (omental) white adipose depots were obtained from male and female cynomolgus monkeys (5–7 yr old, weighing 2.8–3.5 kg). The animals were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were anesthetized with pentobarbital before tissue dissection. Adipocytes were isolated from the specimens by collagenase digestion, as described by Rodbell (23). The fat was placed in buffer 1 [Krebs-Ringer bicarbonate buffer with 5% albumin, 6 mmol/L glucose, 0.1 mmol/L ascorbic acid, 10 nmol/L N6-phenylisopropyladenosine (PIA), and 200 nmol/L adenosine, adjusted to pH 7.4] at 37 C. Each tissue was minced, and adipocytes were isolated by incubation in buffer 1 containing collagenase (3 mg/g tissue) with gentle shaking at 37 C for 1 h. Cells were filtered through nylon, washed twice with 25 mL buffer 1, and spun briefly in a clinical centrifuge (setting 3 for 15 s, no brake). The infranatant buffer was discarded, and the cells were washed three times in 5–10 vol buffer 1. Cell morphology was visualized using a Nikon Microphot-FXA microscope (x20 objective; Nikon, Melville, NY).
Lipolysis assay
In each assay, 100 mg packed cells were added per mL buffer 2 (same as buffer 1, but adenosine was replaced by adenosine deaminase at 0.5 U/mL) in a 4-mL polypropylene tube. Cell treatments (denopamine, procaterol, or CGP12177A in the absence or presence of bupranolol) were prepared approximately 1 h before the experiment and were warmed to 37 C for 5 min before the addition of cells. Cells were incubated for 90 min at 37 C with vigorous shaking; the assay tubes were then placed on ice, and 350 µL infranatant were isolated from each tube and placed in 1.5-mL Eppendorf tubes. The infranatant was spun at 12,000 x g for 20 s, and 150 µL infranatant were separated. Glycerol was determined enzymatically using a Roche COBAS-MIRA S clinical analyzer (Roche Diagnosis, Blackburg, NJ) and reagent from Miles (Tarrytown, NY).
Oxygen consumption assay
A 100-mg aliquot of axillary brown adipocytes was added to 3 mL Krebs-Ringer bicarbonate buffer containing 5% BSA, 6 mmol/L glucose, 0.1 mmol/L ascorbic acid, and 10 nmol/L PIA; saturated with 95% oxygen-5% carbon dioxide; and adjusted to pH 7.4. The assay was performed in presiliconized chambers with stirring to allow complete suspension of the cells using a Yellow Springs Instrument Co. model 5300 Biological Oxygen Monitoring System (Yellow Springs, OH) and a standard 5331 oxygen probe. The cells were equilibrated to 37 C for 5 min, and basal levels of oxygen consumption were measured at 1-min intervals over a 5-min period. Compounds (denopamine, procaterol, and CGP12177A, in the absence or presence of bupranolol) were added through an access port, and the response was measured at 1-min intervals over the following 5–8 min. Agonist-mediated effects were calculated relative to the basal level in the same cells.
RNA analysis
RNA was isolated by a modification of the procedure of Chomczynski and Sacchi (24). Adipose tissue lipid was extracted by the addition of 2 vol chloroform-isoamyl alcohol (49:1). Poly(A)+ RNA was isolated using oligo(deoxythymidine) cellulose and was used in the reverse transcription-PCR (RT-PCR) protocol and was also electrophoresed on a 1.5% agarose-0.66 mol/L formaldelhyde denaturing gel in a 3-(N-morph-olino)propanesulfonic acid buffering system. RNA was transferred to nylon membrane (Hybond-N, Amersham, Arlington Heights, IL) and UV cross-linked. The blots were probed with 32P random primer-labeled PCR product. Prehybridized blots were hybridized overnight at 42 C in the presence of 50% formamide, washed at high stringency, and then exposed overnight against Kodak X-Omat x-ray film (Eastman Kodak, Rochester, NY).
RT-PCR and sequencing
RT-PCR was performed using the GeneAmp RNA PCR Kit (part no. N808-0017, Perkin-Elmer, Norwalk, CT). The RT-PCR product was inserted into a Novagens pT7Blue T-Vector (catalogue no. 69820–1, Novagen, Madison, WI) Sequence analysis was performed using an Applied Biosystems (Foster City, CA) 373 Automated DNA Sequencer. The primers used in sequencing were supplied with the Novagens pT7Blue-Vector Kit (catalogue no. 69837). Dye-labeled dideoxynucleotide sequencing was performed using the PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit (part no. 401384, Applied Biosystems, Foster City, CA).
Membrane binding assays
CHO cells that were transfected with human ß1-, ß2-, or ß3-AR were obtained from Dr. A. D. Strosberg (25). Cells (between passages 22 and 68), were grown to confluence in Ham’s F-12 medium (with 2 mmol/L glutamine, without glycine, hypoxanthine, or thymidine) containing 10% heat-inactivated FCS. Cells were harvested in Ham’s F-12 medium and homogenized in 50 mmol/L Tris-HCl (pH 7.4), 1 mmol/L ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 10 mmol/L MgCl2, 1 mmol/L phenylmethylsulfonylfluoride, and 0.24 U aprotinin using a Brinkmann Polytron (setting 7, three times, 10 s each time; Westbury, NY). The homogenate was passed through cheesecloth and centrifuged at 40,000 x g for 15 min at 4 C. The membrane pellet was resuspended in buffer and washed three times by centrifugation at 40,000 x g for 15 min. The final pellet was resuspended in homogenization buffer and frozen at -80 C.
[125I]Iodocyanopindolol binding
Assays were conducted in a 250-µL volume containing 100 µL cell membranes, 100 µL [125I]iodocyanopindolol (50 pmol/L for CHO-ß1, 25 pmol/L for CHO-ß2, and 250 pmol/L for CHO-ß3), 50 µL displacing drug, or 2 µmol/L (±)propranolol to define nonspecific binding to CHO-ß1 and CHO-ß2 cell membranes or 1 µmol/L bucindolol for CHO- ß3 cell membranes, prepared in buffer [50 mmol/L Tris-HCl (pH 7.4) and 5 mmol/L MgCl2]. Drugs were dissolved in 100% dimethylsulfoxide before dilution in buffer. The binding reaction was started by the addition of membranes, and the tubes were incubated with shaking at room temperature for 2 h (for CHO-ß1 and CHO-ß2 cell membranes) or 30 min (for CHO-ß3 cell membranes). Bound and free radioligand were separated by filtration on a Tomtec cell harvester (Tomtec, Hamden, CT) (26). Membrane protein was determined using BCA reagent (Pierce Chemical Co., Rockford, IL), with BSA as standard. Binding data were analyzed by iterative fitting to a one-site model, and inhibition constants (Ki) were calculated from IC50 values using the equation of Cheng and Prusoff (27).
Reagents
[125I]Iodocyanopindolol CYP (1900–2200 Ci/mmol) was purchased from Amersham (Arlington Heights, IL). (-)Isoproterenol bitartrate and procaterol were obtained from Sigma Chemical Co. (St. Louis, MO). CGP12177A was purchased from RBI (Natick, MA). Bupranolol was provided by Schwarz Pharma (Monheim, Germany), denopamine was obtained from Toshe (Tokyo, Japan), and ZD2079 was a gift from Zeneca Pharmaceuticals (London, UK).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tissue isolated from the axillary depot was visually distinct from
that from the omental depot, in that it was brown in appearance and
resembled rodent BAT, whereas omental fat was yellow. When viewed
microscopically, adipocytes isolated from the axillary depot had a
multilocular appearance, characteristic of brown adipocytes isolated
from rodents (Fig. 1
). To verify that this tissue
contained UCP, a fragment of the cynomolgus UCP message was cloned
using RT-PCR. Primers were developed by comparing known sequences for
homologous regions of UCP sequence. These primers were used in the
RT-PCR reaction to produce an expected product of 728 bp. This PCR
product was then cloned into the pT7-blue vector (Novagen) and later
sequenced. The sequence was compared to known sequences using BLAST
(28), and the highest scores were given to known UCP sequences. Human
sequences had the greatest homology (95.8%) to the cynomolgus monkey
sequence (Fig. 2). The PCR product obtained from the
RT-PCR reaction was used to screen polyadenylated
[poly(A)+] RNA from both abdominal (WAT) and axillary
(BAT) adipose tissue. Northern blot analysis indicated a 1.6-kilobase
transcript in samples from both tissues, indicating the presence of UCP
mRNA in both depots. However, the level of UCP mRNA was severalfold
higher in the axillary sample than in the abdominal sample (Fig. 3).
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Denopamine (ß1-AR agonist), procaterol
(ß2-AR agonist), and CGP12177A (ß3-AR
agonist, ß1- and ß2-AR antagonist; Table 1) were used to study the stimulation of lipolysis by
each ß-AR subtype in isolated abdominal white and axillary brown
adipocytes and the stimulation of oxygen consumption in axillary brown
adipocytes. Isoproterenol was used as a full, nonselective ß-AR
agonist in all experiments.
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The dose-response curves for ß-AR stimulation of lipolysis in
white adipocytes are shown in Fig. 4A. The
EC50 for the lipolytic response to isoproterenol was 0.48
nmol/L. All other measurements of intrinsic activity (IA) were based on
the isoproterenol maxima. Denopamine and procaterol were full agonists
(IA = 98% and 95%), with EC50 values of 200 and 0.8
nmol/L, respectively. CGP12177A was a partial agonist (IA = 78%),
with an EC50 of 55 nmol/L. Total cellular lipolysis in
brown adipocytes (Fig. 4B) was lower than that in white adipocytes
(Fig. 4A). The maximal isoproterenol-mediated lipolysis was 1.2
µmol/100 mg cells·90 min in white adipocytes and 0.4 µmol/100 mg
cells·90 min in brown adipocytes. The EC50 of
isoproterenol for the lipolytic response in brown adipocytes was 4
nmol/L. Denopamine had an EC50 of 500 nmol/L (IA =
74%), procaterol had an EC50 of 83 nmol/L (IA =
59%), and CGP12177A had an EC50 of 1.6 µmol/L (IA =
62%). The dose-response curves for all agonists, with the exception of
denopamine, were shallow. Thus, EC50 values for agonists
were generally higher in brown than in white adipocytes.
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Bupranolol, a potent ß1- and ß2-AR
antagonist and weaker ß3-AR antagonist (Table 1
), was
used to determine the concentration of bupranolol at which the
ß1-AR and ß2-AR components of lipolysis
were eliminated while maintaining a ß3-AR-mediated
lipolytic response. Brown adipocytes were incubated with agonists
(isoproterenol, denopamine, procaterol, and CGP12177A) at a
concentration that elicited 80% of the maximal lipolytic response in
the presence of increasing concentrations of bupranolol.
ß1- and ß2-AR-mediated lipolysis was
completely abolished at a concentration of 10 µmol/L. The
ß3-AR-mediated response was maintained at a maximal
response at this concentration and was not abolished until the
concentration of bupranolol reached 1 mmol/L (Fig. 5).
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Isoproterenol, denopamine, procaterol, and CGP12177A were
used to study the ß-AR stimulation of oxygen consumption in isolated
axillary brown adipocytes. A dose response for the stimulation of
oxygen consumption by isoproterenol is shown in Fig. 6.
Adipocytes were incubated with denopamine, procaterol, and CGP12177A at
a concentration of 100 µmol/L, as significant stimulation of oxygen
consumption could not be obtained during the time course of these
experiments (30 min) at concentrations less then 100 µmol/L.
Isoproterenol, denopamine, and procaterol demonstrated an increase in
oxygen consumption of approximately 75–80% over the basal value (Fig. 7), although the stimulation with procaterol was not
statistically significant. CGP12177A stimulated oxygen consumption by
approximately 35% over the basal value. In the presence of 10 µmol/L
bupranolol, there was complete inhibition of oxygen consumption by
denopamine and procaterol; however, the stimulation by CGP12177A was
not affected.
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As BRL37344 (29) and ZD2079 (30) have been shown to be potent ß3-AR agonists in rodents and stimulate oxygen consumption in rodent BAT, the effects of these compounds in primary cynomolgus brown adipocytes were evaluated. There was no increase in oxygen consumption rates above the basal level after incubation with 100 µmol/L BRL37344 or ZD2079 (data not shown), which is consistent with the minimal ß3-AR activity of both compounds when evaluated in clinical studies.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Northern blot analysis of abdominal WAT and axillary BAT indicated that a greater amount of UCP mRNA was seen in the axillary brown adipose depot. A small amount of UCP mRNA was also detectable in the abdominal adipose depot, which morphologically resembled white adipocytes. Recently, the presence of UCP has been detected in baboon axillary adipose tissue as well as perirenal, pericardial, periaortic, popliteal, and abdominal fat (11). These results indicate that clusters of brown adipocytes exist within white adipose depots in nonhuman primates.
The pharmacological characterization of the ß-AR subtypes in stimulating lipolysis and oxygen consumption in cynomolgus brown adipocytes was evaluated. The primary difficulty in studying the responses of the different ß-AR subtypes is that all are G protein-coupled activators and produce a similar result: specifically, a lipolytic response after agonist activation. Experiments were designed to block the effects of a selective ß1- or ß2-AR agonist while maintaining ß3-AR agonist activity. This was accomplished by using bupranolol, which is a strong ß1- and ß2-AR antagonist and a weak ß3-AR antagonist. Although CGP12177A has been reported to be a ß1-AR agonist in transfected cells expressing high receptor densities (31), the use of 10 µmol/L bupranolol would block any effect of CGP12177A at the ß1-AR and would provide the ß3-AR-mediated stimulation of functional activity in adipocytes.
The studies using specific ß1-, ß2-, and ß3-AR agonists indicated that stimulation at each ß-AR elicits a lipolytic response. The rank order of potency of the agonists in white adipocytes was isoproterenol > procaterol > denopamine > CGP12177A. This order of potency is similar to that reported in other studies using nonhuman primate and human white adipocytes (8). Comparison of the lipolytic response elicited by isoproterenol in brown adipocytes with that in white adipocytes indicated that the intrinsic activity was substantially lower in brown adipocytes. Moreover, the maximal responses of denopamine and procaterol were less in brown than in white adipocytes. The reason for these differences may relate to receptor number or proportion of ß-AR subtypes. The ß2-AR-selective agonist procaterol was a highly effective agonist in white adipocytes, and maximal lipolytic responses were similar to those using isoproterenol. These data suggest that lipolysis in white adipocytes had a major ß2-AR component. However, in brown adipocytes, procaterol was a less effective lipolytic agent, and maximal responses were only 50–60% of the isoproterenol maximum. All agonists had lower overall potency for stimulating lipolysis in brown vs. white adipocytes, and EC50 values were shifted 2.5-fold for denopamine and 103-fold for procaterol. These differences may relate to differences in the receptor number or coupling efficiency of the ß-AR subtypes.
To further evaluate the physiological response to ß-AR agonists in brown adipocytes, the effects of isoproterenol, denopamine, procaterol, and CGP12177A on oxygen consumption in axillary brown adipocytes were determined. At a concentration of 100 µmol/L, isoproterenol, denopamine, and procaterol produced an equal stimulatory response of an approximately 75–80% change in oxygen consumption over basal levels. CGP12177A produced approximately a 35% change in oxygen consumption over basal levels. Furthermore, in the presence of a concentration of bupranolol that blocked stimulation by the ß1- and ß2-ARs, the effect of CGP12177A was maintained, indicating that ß3-AR mediates the stimulation of oxygen consumption in cynomolgus brown adipocytes.
The requirement for such a high level (i.e. 100 µmol/L) of ß-agonists used in this study to stimulate oxygen consumption in brown adipocytes suggests that these cells, which were isolated from adult nonhuman primates housed at room temperature, may be dormant and, therefore, less responsive than brown adipocytes isolated from rodents. Alternatively, the 30-min time frame used in this study (to maintain cell viability under stirring conditions) may have been submaximal for UCP-mediated stimulation of oxygen consumption, contributing to the relative insensitivity to ß-AR agonists.
These results demonstrate that each ß-AR subtype could stimulate oxygen consumption and lipolysis in brown adipocytes and show that the ß3-AR has functional activity in this nonhuman primate species. However, a recent study using BAT from baboons failed to detect a ß3-AR component of lipolysis (11). This may be attributed to the use of tissue fragments rather than isolated brown adipocytes, as used in the present study. The low level of ß3-AR activity may not have been detected when measured in heterogeneous tissue, which contains connective and vascular tissue in addition to adipose tissue. The use of isolated cells also has its limitations, in that adenosine and PIA are used to maintain a low basal lipolytic rate during cell isolation, which then requires the addition of adenosine deaminase to achieve a significant lipolytic effect after treatment with ß-AR agonists.
Another explanation for the discrepancy between this study and that in baboons (11) is that species differences may exist, and cynomolgus monkeys may express a higher level of ß3-AR activity than do baboons. It is possible that expression of ß3-AR activity in BAT may be inversely related to the size or age of the animals and, therefore, is lower in larger older baboons (although the cynomolgus monkeys were mature adults, and the ages of the baboons were not reported). Thus, additional studies are necessary to identify the most appropriate model for human BAT metabolism and the effect of age, body size, and possibly gender on ß3-AR expression in adipocytes.
The functional role of the ß3-AR in nonhuman primate and human adipose tissue is still controversial. This is in part due to the low level and variability of expression of this receptor in these species as well as the modest selectivity of the pharmacological agents used. We have demonstrated in this study that all three ß-AR subtypes can stimulate brown adipocyte lipolysis and oxygen consumption. Further studies of BAT function in humans is essential to determine the role of the ß3-AR in human metabolism. As a good cell model to study human brown adipose tissue function is not available, and rodent model systems may not be analogous to humans, primary brown adipocytes from cynomolgus monkeys may be an appropriate surrogate model.
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Acknowledgments |
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Received July 11, 1996.
Revised September 30, 1996.
Accepted October 14, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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