(Received for publication, November 22, 1996, and in revised form, March 27, 1997)
From the Division of Endocrinology, Department of
Medicine, Beth Israel Deaconess Medical Center and Harvard Medical
School, Boston, Massachusetts 02215, the ¶ Department of
Biochemistry, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5,
and the
Diabetes and Metabolism Unit, Evans Department of
Medicine, Department of Biochemistry, Boston University Medical Center,
Boston, Massachusetts 02118
3-Adrenergic receptors (
3-ARs) are
expressed predominantly on white and brown adipocytes, and acute
treatment of mice with CL 316,243, a potent and highly selective
3-AR agonist, produces a 2-fold increase in energy expenditure, a
50-100-fold increase in insulin levels, and a 40-50% reduction in
food intake. Recently, we generated gene knockout mice lacking
functional
3-ARs and demonstrated that each of these responses were
mediated exclusively by
3-ARs. However, the tissue site responsible
for producing these actions is unknown. In the present study,
genetically engineered mice were created in which
3-ARs are
expressed exclusively in white and brown adipocytes (WAT+BAT-mice), or
in brown adipocytes only (BAT-mice). This was accomplished by injecting
tissue-specific
3-AR transgenic constructs into mouse zygotes
homozygous for the
3-AR knockout allele. Control, knockout, WAT+BAT,
and BAT-mice were then treated acutely with CL, and the effects on
various parameters were assessed. As previously observed, all effects of CL were completely absent in gene knockout mice lacking
3-ARs. The effects on O2 consumption, insulin secretion, and
food intake were completely rescued with transgenic re-expression of
3-ARs in white and brown adipocytes (WAT+BAT-mice), demonstrating
that each of these responses is mediated exclusively by
3-ARs in
white and/or brown adipocytes, and that
3-ARs in other tissue sites were not required. Importantly, transgenic re-expression of
3-ARs in
brown adipocytes only (BAT-mice) failed to rescue, in any way, CL-mediated effects on insulin levels and food intake and only minimally restored effects on oxygen consumption, indicating that any
effect on insulin secretion and food intake, and a full stimulation of
oxygen consumption required the presence of
3-ARs in white adipocytes. The mechanisms by which
3-AR agonist stimulation of
white adipocytes produces these responses are unknown but may involve
novel mediators not previously known to effect these processes.
Obesity is a prevalent condition frequently associated with
diabetes, hypertension, and cardiovascular disease. Because available treatments are minimally effective, substantial efforts have been directed toward the discovery of new, effective, anti-obesity drugs.
The 3-adrenergic receptor (
3-AR)1
represents one of a number of potential anti-obesity drug targets for
which selective agonists have been developed (1-3). The
3-AR is
encoded by a distinct gene that is expressed predominantly in white and
brown adipocytes (4-7), important sites for energy storage and energy
expenditure, respectively. Selective activation of
3-ARs leads to
marked increases in triglyceride breakdown (lipolysis) and energy
expenditure (1-3), and long term treatment of obese rodents with
3-selective agonists reduces fat stores and improves obesity-induced
insulin resistance (1-3). Thus,
3-selective agonists are promising
anti-obesity compounds.
Acute treatment of rodents with 3-selective agonists causes a number
of diverse metabolic effects including a 2-fold increase in energy
expenditure as measured by effects on oxygen consumption, a
10-100-fold increase in insulin levels, and a 40-50% reduction in
food intake (8-11). While the effects on lipolysis and energy expenditure are likely to be mediated by
3-ARs, uncertainty has existed regarding the identity of receptors mediating the acute effects
on insulin levels and food intake. Recently, we generated gene knockout
mice lacking functional
3-ARs (11). These animals have a slight
increase in body fat; but otherwise, they appear to be normal, probably
because of adaptations which compensate for the absence of
3-ARs,
one example being the observed up-regulation of
1-AR gene expression
in brown and white adipose tissue (11). Of significance, these mutant
mice are completely resistant to the ability of CL 316,243 (1), a
3-selective agonist, to increase lipolysis, energy expenditure,
insulin levels, and to reduce food intake (11). Thus, each of these
effects is mediated exclusively by
3-ARs.
The relative role of 3-ARs in white versus brown
adipocytes, as well as
3-ARs in other sites, in mediating each of
these responses is also unresolved. For example, is the marked
stimulatory effect of
3-selective agonists on energy expenditure
mediated exclusively by stimulation of brown adipocytes, or must white adipocytes also be stimulated to supply free fatty acids as fuel for
brown adipocytes? Furthermore, as
3-ARs have been reported to exist
in the gastrointestinal tract, brain, heart, and prostate and since
other sites of expression cannot be excluded (5, 12-15), is it
possible that receptors in non-adipocyte locations mediate some or all
of the effects on energy expenditure, insulin secretion, and food
intake? At present, the answers to questions such as these are
unknown.
In general, it has been difficult to determine the relative role of
various target tissues in mediating complex physiologic responses.
Genetic engineering in mice, however, provides a means by which these
issues can be addressed. In the present study, we have combined gene
knockout and transgenic techniques to create mice in which functional
3-ARs are completely absent (knockout mice; see Ref. 11), or are
expressed exclusively in selected tissues, namely white and brown
adipose tissue (WAT+BAT-mice) or brown adipose tissue only (BAT-mice).
To create WAT+BAT-mice and BAT-mice, transgenic constructs were
generated in which murine
3-AR gene expression is driven by the
tissue-specific promoter/enhancers, aP2 for white and brown adipose
tissue expression (16) and UCP for brown adipose tissue expression (17,
18). These transgenic constructs were then injected into fertilized
mouse zygotes homozygous for the
3-AR gene knockout allele (11),
thus creating mice in which functional
3-ARs are restricted to white
and brown fat (WAT+BAT-mice), or brown fat only (BAT-mice). Control,
knockout, WAT+BAT-, and BAT-mice were then used to investigate the
relative role of
3-ARs in white versus brown adipose
tissue, as well as
3-ARs in other sites, in mediating a number of
responses to
3-selective agonists.
Partial
restriction enzyme maps for the murine 3-AR wild-type allele, the
3-AR knockout allele (11), the aP2-
3-AR transgene used to create
WAT+BAT-mice, and the UCP-
3-AR transgene used to create BAT-mice are
shown in Fig. 1. The gene knockout allele is notable for its absence of
306 bp of
3-AR coding sequence, NheI to XhoI,
spanning from
3-AR residue 120 in the middle of the third
transmembrane domain to residue 222 in the COOH-terminal end of the
fifth transmembrane domain. The aP2-
3-AR transgene was created by
fusing 5.4 kb of 5
-flanking regulatory sequence of the murine aP2 gene
(16),
5.4 kb (EcoRI) to + 21 bp (PstI), to a
3.0-kb fragment of mouse
3-AR genomic sequence (7, 19), spanning
from 11 bp 5
of the
3-AR start codon (Nar I) to
approximately 240 bp 3
of the
3-AR AATAAA polyadenylation signal
(BglII). The UCP-
3-AR transgene was created by fusing 4.0 kb of 5
-flanking regulatory sequence of the murine UCP gene (17),
3.9 kb (EcoRI) to + 120 bp (PstI), to the mouse
3-AR 3.0-kb fragment described above. The transgenes were excised
from plasmid vector sequence, gel-purified, and then injected into male
pronuclei (20) of zygotes homozygous for the
3-AR knockout allele
(11). To identify transgenic animals, genomic DNA from mouse tails was
digested with BamHI (aP2-
3-AR construct) or
NcoI (UCP-
3-AR construct), electrophoresed, Southern
blotted, and hybridized to an 862-bp
3-AR genomic probe (between 545 bp 5
and 317 bp 3
of the
3-AR ATG).
3-AR knockout mice were originally created on an inbred FVB
background (11). The knockout allele and the WAT+BAT and BAT-transgenes have been maintained on an inbred FVB background. Control animals were
wild-type inbred FVB mice housed under conditions similar to the
genetically modified animals. All animals were housed at 24 °C, had
free access to food (Purina Chow 5008) and water, and were handled in
accordance with the principles and guidelines established by the
National Institutes of Health.
Total RNA was isolated from white and brown
adipose tissue, brain, colon, kidney, liver, and skeletal muscle of
male mice, age 12 to 20 weeks old, using a Brinkmann homogenizer and
RNA STAT-60 solution (Tel-Test "B", Inc., Friendswood, TX). 3-AR mRNA was detected using standard Northern blotting techniques and
20 µg of total RNA. A
3-AR hybridization probe was generated by
random priming from a 306-bp cDNA template corresponding to codons
120-222. Note that this 306-bp cDNA region corresponds to the
NheI to XhoI
3-AR fragment deleted during the
creation of the
3-AR gene knockout allele. The gels were stained
with ethidium bromide, and the abundance of 18 S and 28 S ribosomal bands were used to establish equal loading. To quantitate
3-AR mRNA expression, radioactive signals from all Northern blots were analyzed using a PhosphorImager (Molecular Dynamics, Image Quant software).
Five hundred pancreatic islets were isolated
from 10 normal mice as described previously (21). Total RNA was
extracted using RNA STAT-60 solution and was then subjected to RT-PCR
analysis using mouse 3-AR and actin primer sets (
3-AR sense
primer, CCTAGCTGTCACCAACCCTTT;
3-AR antisense primer,
GACGAAGAGCATCACAAGGAG; actin sense primer, GTGGGCCGCTCTAGGCACCA; actin
antisense primer, CGGTTGGCCTTAGGGTTCAGGGGGG). RNA samples were treated
with DNase prior to performing RT-PCR. The
3-AR primer set amplifies
a 260-bp fragment, and the actin primer set amplifies a 245-bp
fragment. First strand synthesis was performed using the SuperScript
preamplification system procedure (Life Technologies, Inc.,
Gaithersburg, MD) and the antisense primers for
3-AR and actin
listed above. PCR was then performed using TaKaRaEx (Otsu, Shiga,
Japan) Taq polymerase. Manufacturers recommended buffer,
nucleotide, and primer concentrations were employed. The samples were
heated to 94 C for 1.5 min and then cycled for 30 cycles using the
following protocol: 98 °C for 20 s, 60 °C for 1 min, and
68 °C for 1.5 min. The PCR products were then analyzed using agarose
gel electrophoresis and ethidium bromide staining.
Oxygen consumption was measured in female, 16-20-week old control, knockout, WAT+BAT-, and BAT-mice before and after treatment with CL at 1 mg/kg body weight (subcutaneously). Preliminary experiments with control mice indicated that this dose exerted maximum effects on oxygen consumption. Oxygen consumption was measured using computerized equipment that included a 1-liter chamber maintained at 28 °C, an air flow of 500 ml/min (regulated with a mass flowmeter, Brooks Instrument Division, Emerson Electric), and an oxygen analyzer (Beckman Industrial Oxygen Analyzer model 755). Mice were awake and unrestrained for the study. The resting rate of oxygen consumption (basal) was assessed when the mice were curled up and still, usually 1-2 h after they had been placed in the chamber. The animals were then injected with CL and returned to the chamber. After injection of CL, a constant rate of oxygen uptake was assessed when mice were again resting.
Effect of CL on Adenylate Cyclase Activity in Membranes Isolated from Brown Adipose TissueMembranes were isolated from interscapular brown adipose tissue of control, knockout, and BAT-mice, and effects of CL on adenylate cyclase activity were assessed as described previously (11).
Effect of CL on Insulin, Glucose, and Free Fatty Acids (FFAs) LevelsCL (1 mg/kg body weight, intraperitoneal) or saline was injected into female, 8-12-week old control, knockout, WAT+BAT-, and BAT-mice; 15 min later, the animals were quickly sacrificed using a small animal decapitator. Whole blood was collected and analyzed for blood glucose levels (One Touch Blood Glucose Meter, Lifescan, Inc., Milpitas, CA). Serum was then isolated and assayed for FFAs (NEFA C Kit, Wako Pure Chemical Industries, Ltd.) and insulin (Rat Insulin Kit, Linco Research Inc., St. Louis, MO). In a preliminary experiment, control mice were injected with CL, and blood samples were obtained before CL and 5, 10, 15, and 60 min after CL injection. Maximal increases in insulin were observed at the 15-min point.
Effect of CL on Insulin Secretion from Isolated Pancreatic IsletsGroups of 20 islets from control, knockout, and WAT+BAT-mice were pre-incubated for 30 min in 3 mM glucose as described previously (21). The media was removed and then replaced with 1 ml of the following test solutions: 3 mM glucose, 11 mM glucose, or 11 mM glucose plus 1 µM CL. Of note, 11 mM glucose is a submaximal stimulatory concentration of glucose. Twenty min later, the incubation media was removed and assayed for insulin.
Effect of CL on Food IntakeThe effect of a single injection of CL (1 mg/kg body weight, intraperitoneal) on food intake was assessed in male, 8-12-week old control, knockout, WAT+BAT-, and BAT-mice. The mice were housed individually during the study. For three days prior to the day of CL treatment, the mice received daily injections of saline to acclimate them to handling. On the day of study, each experimental group was divided in half, and one half was treated with saline while the other half received CL. Food was weighed before and 24 h after injection, and the weight of food missing was assumed to represent g of food eaten. The cages were inspected for food spillage and none was noted.
A large colony of homozygous
3-AR knockout mice was established (11) and used to generate
homozygous
3-AR knockout zygotes. These zygotes were injected with
the aP2-
3-AR or UCP-
3-AR transgenes (Fig. 1) to
create WAT+BAT-mice and BAT-mice, respectively. For the aP2-
3-AR
construct, nine founders were produced, six of which transmitted the
transgene to offspring. Of these six transmitting WAT+BAT-lines, five
lines expressed
3-AR mRNA in white and brown adipose tissue, and
three of these expressing lines were selected for further analyses
(WAT+BAT-1, WAT+BAT-2, and WAT+BAT-3). For the UCP-
3-AR construct,
six founders were produced, five of which transmitted the transgene to
offspring. Of these five transmitting BAT-lines, four lines expressed
3-AR mRNA in brown adipose tissue, and two of these expressing
lines were selected for further analyses (BAT-1 and BAT-2).
Northern blotting was used to determine the
expression level of 3-AR mRNA. The probe used for these studies
corresponded to the 306-bp
3-AR coding sequence segment, which was
deleted during the creation of the knockout allele (Fig. 1).
Consequently, no signal was detected in RNA samples isolated from
homozygous
3-AR knockout mice (Fig. 2). As expected,
WAT+BAT-lines expressed
3-AR mRNA in white and brown adipose
tissue while BAT-lines expressed
3-AR mRNA in brown adipose
tissue only. With respect to white adipose tissue, line WAT+BAT-1
expressed at a level that was 44% above control mice while lines
WAT+BAT-2 and WAT+BAT-3 expressed at levels that were 14 and 47% below
control mice, respectively. With respect to brown adipose tissue, lines
WAT+BAT-2 and WAT+BAT-3 expressed at levels that were approximately
2.5-fold higher than control mice, lines WAT+BAT-1 and BAT-1 expressed
at levels that were just slightly above controls, while line BAT-2
expressed at a level that was slightly below control mice.
Northern blot analysis of total RNA isolated from brain, colon, kidney,
liver, and skeletal muscle demonstrated that 3-AR mRNA
expression, with the possible exception noted below, was restricted to
adipose tissue (Fig. 3). In the case of BAT-lines, no
expression was detected outside of brown adipose tissue (no signal in
white adipose tissue, brain, colon, kidney, liver, and skeletal
muscle). In the case of WAT+BAT-lines, however, low-level signals were
occasionally detected in skeletal muscle but not in brain, colon,
kidney, or liver. It is difficult to establish whether this apparent
expression in skeletal muscle was due to ectopic expression in myocytes
or represents expression in adipocytes that are resident within or
surrounding this tissue. To address this issue, we carefully dissected
away visible fat from skeletal muscle obtained from line WAT+BAT-1.
Northern blot analysis of these RNA samples failed to detect any
specific
3-AR mRNA signal, indicating that the weak signal
observed in muscle samples from this line were likely due to the
presence of adipocytes.
Effect of CL on Oxygen Consumption
Acute treatment with CL
increased oxygen consumption in control mice by 89% (Fig.
4). As was previously observed (11), this response was
completely absent in 3-AR knockout mice (increase of 0%). In
WAT+BAT-lines, the effect of CL on oxygen consumption was restored,
with lines WAT+BAT-1 (increase of 120%) and WAT+BAT-2 (increase of
103%) having responses that were equal to or greater than control mice
and line WAT+BAT-3 (increase of 60%) having a response that was
somewhat less than control mice. CL also increased oxygen consumption
in BAT-1 (increase of 24%) and BAT-2 (increase of 17%) transgenic
mice; however, the O2 consumption response in these mice
was notably less than that observed in control mice and in all three
lines of WAT+BAT-transgenic mice.
Effect of CL on Adenylate Cyclase Activity in Membranes Isolated from Brown Adipose Tissue
Previously, it was assumed that the
O2 consumption response to CL was mediated exclusively by
3-ARs in brown adipocytes. Therefore, the markedly reduced
O2 consumption response of BAT-1 and BAT-2 mice was
somewhat unexpected, especially since Northern blot expression data
(Fig. 2) demonstrated that BAT-1 and BAT-2 mice have normal amounts of
3-AR mRNA in brown fat. However, it is formally possible that
the mRNA transcribed from the UCP-
3-AR transgene bears an
unknown mutation created during transgene construction or that the
transgene-derived RNA transcript is translated at a lower efficiency
due to differences in 5
-untranslated sequence. To rule out these
important possibilities, we assayed for functional
3-ARs by
assessing CL-mediated stimulation of adenylate cyclase activity in
membranes isolated from interscapular brown adipose tissue of control,
knockout, BAT-1, and BAT-2 mice. As previously reported (11), adenylate
cyclase activity was not stimulated in membranes derived from knockout
mice (Fig. 5). In contrast, adenylate cyclase activity
was stimulated normally in membranes derived from BAT-1 and BAT-2 mice,
demonstrating that BAT-mice express normal amounts of functional
3-ARs in brown adipose tissue and that the reduced O2
consumption response in these mice must be due to the absence of
3-ARs in white adipose tissue.
Effect of CL on Insulin, Glucose, and FFAs Levels
Previous
studies have demonstrated that acute treatment of rodents with 3-AR
selective agonists increases serum insulin levels by 10-100-fold (8,
11). To establish the time course for this effect, as well as for the
effects on FFA and glucose levels, an initial experiment was performed
on control mice (Fig. 6). This study demonstrated that
FFA levels increased by 4.4-fold, with a near maximal increase being
observed as early as 5 min. Insulin increased by 50-fold with a near
maximal increase being observed at the 10-min point. A substantially
smaller increase in insulin was observed at the 5-min point. Glucose
concentrations decreased by 58%, with a near maximal decrease being
observed at the 10-min point. At the 60-min point, insulin levels fell slightly but were still markedly elevated (increased by 24-fold) despite the presence of hypoglycemia during the preceding 50 min. The
large increase in insulin levels and the persistent elevation despite hypoglycemia indicate that the stimulus for insulin secretion was potent. In all further experiments, FFAs, insulin and glucose were assessed at the 15-min point.
As previously reported (11), the ability of CL to increase FFAs and
insulin and to reduce glucose was absent in 3-AR knockout mice (Fig.
7). These responses were completely restored in
WAT+BAT-1 mice (FFAs increased by 3.6-fold, insulin increased by
91-fold, and glucose decreased by 55%), were only partially restored
in WAT+BAT-2 mice (FFAs increased by 2.7-fold, insulin increased by
13-fold, and glucose decreased by 49%), and were minimally restored in
WAT+BAT-3 mice (FFAs increased by 1.6-fold, insulin increased by
3.7-fold, and glucose decreased by 13%). Of note, as previously shown
in Fig. 2, left panel,
3-AR mRNA expression in white
adipose tissue was reduced in WAT+BAT-2 and WAT+BAT-3 mice, with the
lowest level of expression being observed in line 3. Thus, it seems
likely that decreased
3-AR gene expression in white adipose tissue
accounts for the reduced responsiveness of these two lines. In BAT
transgenic mice (BAT-1 and BAT-2), no effect of CL was observed on
FFAs, insulin, and glucose, supporting the idea that
3-ARs in white
adipose tissue are required for these responses.
Assessment of
Total RNA was extracted from control mouse
pancreatic islets and was then assessed for 3-AR and actin mRNA
expression using RT-PCR (data not shown). Total RNA from white adipose
tissue was used as a positive control.
3-AR mRNA was not
detected in RNA samples isolated from pancreatic islets but was
detected in RNA samples derived from white fat. In contrast, actin
mRNA was detected in both islet and white adipose tissue RNA
samples. Thus, mouse pancreatic islets appear not to express detectable
levels of
3-AR mRNA.
To rule out any possibility that CL stimulated insulin
secretion via a direct effect on pancreatic -cells, islets were
isolated from control, knockout, and WAT+BAT mice and then treated with CL in the presence of submaximal stimulatory concentrations of glucose
(11 mM). The secretory response at 11 mM
glucose is one third to half of that obtained upon addition of arginine
or an increase in glucose to 20 mM (21), demonstrating
the sensitivity of this system for further stimulation of insulin
secretion. As shown in Fig. 8, CL had no effect on
insulin secretion in any group. This is in agreement with a previous
study of isolated islets (8). Thus, the stimulatory effect of CL on
insulin secretion is not mediated via stimulation of
3-ARs within
islets.
Effect of CL on Food Intake
Acute treatment of control mice
with CL reduced food intake by 35-45% (Fig. 9,
right and left panels). As was previously
observed (11), this effect was completely absent in 3-AR knockout
mice. Significantly, the CL-mediated inhibition of food intake was
observed in all WAT+BAT-transgenic mice (WAT+BAT-1, 89% inhibition;
WAT+BAT-2, 94% inhibition; WAT+BAT-3, 63% inhibition). Of note, the
degree of inhibition observed in each WAT+BAT transgenic line was
greater than that observed in control mice. In contrast to
WAT+BAT-transgenic mice, food intake was not inhibited in
BAT-transgenic mice (BAT-1 or BAT-2 mice), consistent with the idea
that
3-ARs in white adipose tissue are required for the effect on
food intake.
3-AR mRNA is expressed predominately in white and brown
adipose tissue (4-7), and
3-selective agonists are potential
anti-obesity drugs (1-3). Chronic treatment of obese rodents with
3-selective agonists decreases fat stores and improves
obesity-linked insulin resistance. Acute treatment of rodents causes a
number of diverse metabolic effects including an increase in oxygen
consumption and insulin levels, and a decrease in food intake. The role
of
3-ARs, and
3-ARs in white versus brown adipocytes
in particular, in mediating each of these responses has been unclear.
This is especially true given (a) the diverse nature of
these responses, (b) the lack of clear explanations as to
how stimulated adipocyte
3-ARs might control insulin secretion and
food intake, and (c) the recent observations suggesting the
presence of
3-ARs in non-adipocyte sites (5, 12-15). Recently, we
generated gene knockout mice that lack functional
3-ARs (11). These
animals are completely resistant to CL-mediated effects on oxygen
consumption, insulin secretion, and food intake demonstrating that each
of these responses are mediated exclusively by
3-ARs (11). To
determine the role of white and brown adipocyte
3-ARs in mediating
these responses, we have combined gene knockout and transgenic
techniques to create mice in which functional
3-ARs are expressed
selectively in white and brown adipocytes (WAT+BAT-mice) or in brown
adipocytes only (BAT-mice). This was accomplished by injecting
tissue-specific
3-AR transgenic constructs (aP2-
3-AR for
WAT+BAT-mice and UCP-
3-AR for BAT-mice) into mouse zygotes
homozygous for the
3-AR knockout allele (11). We then treated
control, knockout, WAT+BAT, and BAT-mice with CL (1), the
3-selective agonist, and determined effects on oxygen consumption,
insulin secretion, and food intake.
The significant findings of this study are that transgenic
re-expression of 3-ARs in white and brown adipose tissue
(WAT+BAT-mice) completely rescued CL-mediated effects on oxygen
consumption, insulin levels,and food intake. Thus, each of these
responses are mediated exclusively by
3-ARs in white and/or brown
adipocytes; receptors in non-adipocyte locations are not required.
Importantly, transgenic re-expression of
3-ARs in brown adipocytes
only (BAT-mice) failed to rescue in any way CL-mediated effects on
insulin levels and food intake and only minimally restored effects on
oxygen consumption, indicating that any effect on insulin secretion and food intake and a full response on oxygen consumption requires the
presence of
3-ARs in white adipocytes. Whether some or all of these
responses require the simultaneous existence of
3-ARs in white and
brown adipocytes or are partially or completely dependent upon
3-ARs
in white adipocytes alone, is not resolved by the present study. In
theory, this question could be addressed in the future by generating
mice that express
3-ARs exclusively in white adipocytes.
It is noteworthy that 3-ARs must be present in both white and brown
adipocytes in order for CL to maximally increase O2
consumption (89% increase in control mice and 60-120% increase in
WAT+BAT-mice). Lack of
3-ARs in white adipocytes significantly
limits the stimulatory effect of CL (17-24% increase in BAT-mice).
The mechanism by which
3-ARs in white adipocytes permit maximal
stimulation of energy expenditure is unknown but might possibly be
related to their role in mobilizing FFAs from triglyceride stores in
white adipocytes, which are then used as fuel for energy expenditure by
brown adipocytes or possibly by some other tissue site. Alternatively,
it is also possible that energy expenditure in white adipocytes becomes
significant following stimulation by CL, an effect not previously
appreciated. Such an effect could be mediated by uncoupling protein-2
(UCP2), a recently identified homologue of UCP1 that is abundantly
expressed in white adipocytes (22). Prior studies have demonstrated
that FFAs are potent activators of UCP1 (23), and based upon the marked
amino acid similarity between UCP2 and UCP1, the same is expected to be
true for UCP2. Thus, a CL-induced increase white adipocyte
intracellular FFA should activate the uncoupling activity of UCP2,
thereby increasing energy expenditure in white fat cells. Future
studies will be needed to define the role of UCP2 in mediating
3-agonist-induced energy expenditure in white adipocytes.
Nevertheless, the observation that white adipocyte
3-ARs are
required for maximal stimulation of energy expenditure may have
important implications for the treatment of human obesity. In humans,
3-ARs are abundant in brown adipocytes (12, 24-26), however, unlike
rodents,
3-ARs in human white adipocytes are either rare or absent
(26-28). Given this and the findings of the present study, it is
reasonable to anticipate that the acute stimulatory effect of
3-agonists on energy expenditure in humans will be less than might
have been expected. Whether this will translate into reduced
anti-obesity effectiveness under chronic treatment situations is
entirely unknown. It will be important to evaluate this possibility by
comparing the long-term anti-obesity effectiveness of CL in mice with
3-ARs in white and brown fat (control mice and WAT+BAT-mice) with
mice where
3-ARs are restricted to brown adipocytes only
(BAT-mice).
The mechanism by which CL increases insulin secretion is not known.
Nevertheless, the stimulus for insulin secretion is extremely potent as
it causes a 50-100-fold increase in insulin levels, which remain
elevated (24-fold increase) despite the presence of hypoglycemia. This
effect cannot be mediated by direct effects of CL on pancreatic
-cells since pancreatic islets appear not to express detectable
levels of
3-AR mRNA and since insulin secretion is not
stimulated following addition of CL to cultured pancreatic islets.
Instead, the present study demonstrates that the effects of CL on
insulin secretion are mediated by stimulation of
3-ARs on white
adipocytes. Importantly, this observation vividly demonstrates that a
signal or signals emanating from white adipocytes can, directly or
indirectly, profoundly alter
-cell function. The identity of this
signal is unknown but could be a protein, FFAs, or another lipid
product released from fat. However, given that the response occurs
within 10 min, and therefore probably does not involve transcription of
new genes, and since adipocytes are not known to possess storage
granules, the stimulus for insulin secretion may not be an
adipocyte-derived protein. In contrast, it is possible that CL-induced
increases in FFAs (3-4-fold) mediate the effect on insulin release.
Previous studies have demonstrated that FFAs are secretagogues for
insulin secretion (29, 30), and the observed time course in the present
study, i.e. increased FFA levels preceding increased insulin
concentrations, is consistent with this possibility. However, the
magnitude of the observed effect on insulin levels (50-100-fold
increase), and its persistence despite the presence of hypoglycemia,
seems to implicate additional factors as well. This is especially true
since FFAs do not stimulate insulin secretion at low glucose levels,
and starvation-induced increases in FFA concentrations do not lead to
increased insulin levels. Thus, it is plausible that some novel factor
emanating from white adipocytes, possibly some lipid product other than FFAs, directly or indirectly stimulates insulin secretion. Given the
extreme potency of this stimulus, it will be important to identify the
mechanism by which this response occurs.
Similarly, the mechanism by which CL treatment acutely decreases food
intake is unknown. From the present study, it is clear that this
response is mediated by 3-ARs in adipose tissue and requires the
presence of
3-ARs on white adipocytes. The nature of this signal is
also unknown. Leptin is a fat-derived protein that regulates appetite
(31-34); however, it cannot account for this effect since leptin
levels decrease substantially following
3-agonist treatment (35),
and this would be predicted to have the opposite effect on food intake.
Insulin is another factor that has been shown to suppress food intake
(36, 37), and as discussed above, insulin levels rise substantially
following treatment with CL. However, insulin may not be mediating the
suppression of food intake since in WAT+BAT transgenic line 3, a
minimal effect was observed on insulin levels but a substantial effect
was observed on food intake. Heat has long been known to have effects
on regulating appetite (38, 39), and it has been postulated that heat
generation by brown adipose tissue regulates food intake (40). Of note, heat production is predicted to be markedly increased in all lines that
had decreased food intake (control mice and WAT+BAT mice, but not BAT
mice). If heat is the signal mediating the suppression of food intake,
then it is predicted that it requires the presence of
3-ARs in white
as well as brown adipocytes. For this and other reasons, it would be of
interest to create mice that express
3-ARs in white adipocytes only.
Finally, other possible candidates for mediating the effects of CL on
food intake are lipid fuels (FFAs, or ketone bodies) or possibly
another fat-derived protein capable of regulating food intake. Future
work will focus on identifying the mechanism responsible for this
effect.
In summary, acute treatment with 3-selective agonists produces a
number of diverse metabolic effects, including stimulation of
O2 consumption, insulin secretion, and inhibition of food
intake. In the present study, we have used a transgenic and gene
knockout approach to genetically identify the receptor and tissues
responsible for mediating each of these important responses. From this
and an earlier study (11), we can conclude that each of these effects are mediated exclusively by
3-ARs on adipocytes. In
addition,
3-ARs in white adipocytes must be present to obtain any
effect on insulin secretion and food intake and for a full effect on energy expenditure. Determining the mechanisms by which stimulation of
adipocytes produces each of these responses will be the focus of future
studies.
We wish to thank Bruce Spiegelman (The Dana Faber Cancer Institute, Boston, Massachusetts) and Leslie Kozak (The Jackson Laboratory, Bar Harbor, Maine) for providing the aP2 and UCP promoter/enhancers, respectively, and Tom Claus (Lederle Laboratories, Pearle River, New York) for providing CL 316,243.