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Departments of Psychiatry and Behavioral Sciences, and Pharmacology (S.C.), and The Sarah W. Stedman Center for Nutritional Studies, Duke University Medical Center, Durham, North Carolina 27710
Address all correspondence and requests for reprints to: Dr. Sheila Collins, Box 3557, Duke University Medical Center, Durham, North Carolina 27710. E-mail: sco{at}galactose.mc.duke.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Considerable effort has been directed toward understanding the biochemical alterations found in adipocytes from genetically obese rodents or humans. For example, previous studies in genetically obese mice have linked poor epinephrine-stimulated triglyceride metabolism and depressed cAMP responses to obesity (10, 11, 12). However, these earlier studies were conducted before the knowledge that a third ß-adrenergic receptor (ß3AR) exists (13). This third ßAR subtype is expressed predominantly in adipocytes, unlike the ß1AR and ß2AR subtypes, which are broadly distributed in tissues throughout the body. Several genetic and biochemical studies suggest that the ß3AR plays a significant role in adipocyte metabolism. Collins et al. (14) reported that the expression and function of the ß3AR and ß1AR subtypes are significantly depressed in both white and brown adipocytes of the obese (C57BL/6J ob/ob) mouse. More recently, we found essentially identical results in the other major mouse models of congenital obesity, such as diabetic, tubby, and fat mice (Daniel, K. W., E. M. Rohlfs, and S. Collins, manuscript in preparation). The significance of these observations is that pharmacological studies have shown that all three ßAR subtypes participate in the stimulation of lipolysis (15, 16) and are able to activate expression of the mitochondrial uncoupling protein (UCP) in brown adipocytes (17). Thus, it is important to fully understand how all three ßAR subtypes regulate normal adipocyte physiology and, in turn, how each of the receptor subtypes themselves is regulated at the transcriptional and functional levels in the lean and obese states.
The earliest evidence suggesting the existence of a third ßAR subtype
also indicated that such a receptor could have an important role in the
regulation of body composition. These initial observations included
reports that short term treatment of genetically obese
(ob/ob) mice with selective ß3AR agonists
could retard the progression of obesity and normalize elevated plasma
glucose and insulin levels (18, 19, 20). Reversal of dietary obesity has
also been reported in rats after a shortterm (
2-week) treatment with
a ß3AR agonist (21). In the studies we report here, we
examined the effects of chronic treatment with a highly selective
ß3AR agonist, CL316,243, on the development of
diet-induced obesity in B/6J and A/J strains of mice, and on the
expression of the three ßAR subtypes in white and brown adipose
tissues (WAT and BAT, respectively) of these animals. Our results
indicate a striking effect of genetic background on both the
development of obesity in response to high fat feeding and the ability
of ß3AR agonists to prevent it.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Blood was collected at 4-week intervals from individual animals. Samples were obtained via retroorbital sinus puncture in nonanesthetized animals after an 8-h fast. Plasma glucose was determined using a Beckman glucose analyzer 2 (Palo Alto, CA), and plasma insulin levels were measured by double antibody RIA using rat insulin as a standard (Linco Research, St. Louis, MO).
After 16 weeks on the diets, animals were killed by decapitation, and adipose tissues were isolated from the following depots: epididymal, interscapular, sc, and perirenal. Tissues were cleaned and minced finely for preparation of total RNA or plasma membranes.
Isolation and analysis of RNA
Total cellular RNA was prepared by the cesium chloride gradient
method as previously detailed (22). For Northern blot hybridization,
RNA was denatured by the glyoxal procedure, fractionated through 1.2%
agarose gels, and blotted onto Biotrans nylon membranes (ICN, Irvine,
CA) as previously described (23). DNA fragments, which were used as
hybridization probes, were obtained from the following sources. For the
three ßAR subtypes, fragments specific for each receptor were
prepared by PCR as previously described (14). For mitochondrial UCP, a
300-bp BglI fragment, kindly provided by Dr. Leslie P.
Kozak, was used (24). The complementary DNA fragment for the ubiquitous
glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase was obtained
from Clontech (Palo Alto, CA). A rat complementary DNA probe for
cyclophilin was obtained from J. Douglas. Radiolabeled probes were
prepared by nick translation of the purified DNA fragments in the
presence of [32P]deoxy-CTP to a specific activity between
3–7 x 108 dpm/µg DNA. Blots were hybridized and
washed as previously described (23, 25).
Preparation of plasma membranes and adenylyl cyclase assay
Adipose tissue was isolated from each group of animals. The
tissue was pooled and minced, and then plasma membranes were prepared
as previously described (14). Adenylyl cyclase activity in these plasma
membrane preparations was measured using established methods (26), and
the cAMP formed was measured by RIA (27) using a polyclonal antiserum
to cAMP. The ß-agonists (-)-epinephrine, (-)-norepinephrine,
(-)-isoproterenol, and (-)-propranolol were obtained from Sigma
Chemical Co. (St. Louis, MO). CL316,243 was a gift from American
Cyanamid Co. Protein concentrations were determined by the method of
Bradford (28).
Data analysis
Adenylyl cyclase dose-response curves and competition binding
curves were analyzed by least squares nonlinear regression. (Graphpad
Prism, San Diego, CA). The best fit to a one-component vs.
2-component model was determined by an F test, and comparisons between
data sets were made by ANOVA. P < 0.05 was considered
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). This increase in body weight was associated with
adipocyte hypertrophy, hyperplasia, and approximately a 50% increase
in their percentage of total body fat, as determined by carcass
analysis and bomb calorimetry (low fat B/6J, 22.4% body fat; high fat
B/6J, 36.8%; low fat A/J, 24.3%; high fat A/J, 32.5%) (7) (our
unpublished observations). As we previously reported, this differential
weight gain occurred despite the fact that B/6J mice did not consume
more calories than A/J mice (7) and were equally as active (8). When
the selective ß3AR agonist CL316,243 was included in the
high fat diet, A/J mice were completely resistant to the obesity
induced by high fat feeding (Fig. 1A). In fact, they actually weighed
slightly less than low fat fed animals at most time points, despite the
fact that they ate as much as or even slightly more than animals
consuming the high fat diet without CL316,243 (29). In contrast, B/6J
mice appeared to be refractory to the ß3AR agonist, at
least under this dietary regimen (Fig. 1B). Furthermore, mice consuming
the CL316,243 diet were no more active than those not receiving
treatment (data not shown).
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and Table 1 illustrate these findings.
For example, levels of both insulin (Table 1) and glucose (Fig. 2) were
significantly increased in A/J and B/6J mice when they were fed a high
fat diet, but both of these factors were more severely elevated in B/6J
mice, confirming our previous observations (7). However, the presence
of CL316,243 in the diet was associated with significant decreases in
serum insulin and glucose levels in A/J mice throughout the 16-week
treatment period. By contrast, although there seemed to be some
improvement in the levels of both insulin and glucose in B/6J mice
during the first month of treatment with CL316,243, ultimately the
hyperglycemia and hyperinsulinemia resulting from high fat feeding were
not reversed.
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). These results are strikingly similar to our
earlier findings in ob/ob mice (14) and indicate impaired
function of both the ß1AR/ß2AR population
of receptors and the ß3AR receptors. However, in A/J
mice, there was a small, but significant, decrease in
ß-agonist-stimulated adenylyl cyclase activity from WAT (Fig. 4). The level of activity was modestly lower throughout
the entire concentration range examined, and the data obtained using
CL316,243 to stimulate adenylyl cyclase activity suggest that the
function of the ß3AR is also slightly decreased.
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indicates that in obese, high fat-fed, B/6J mice there
was nearly a complete loss in the ability of either norepinephrine or
CL316,243 to stimulate cAMP production in interscapular BAT (IBAT). In
A/J mice (Fig. 6), although there was a significant
reduction in ß-agonist-stimulated adenylyl cyclase activity in the
high fat-fed animals relative to that in the low fat-fed mice, there
was, nevertheless, significant retention of activity compared with that
in the fat-fed obese B/6J mice (compare Figs. 5 and 6). These
differences in adenylyl cyclase activity between the two strains were
also observed in WAT and IBAT of CL316,243-treated animals (Figs. 7 and 8, respectively). In both adipose
depots the ß-agonist-stimulated adenylyl cyclase activity in
membranes from B/6J mice, using either epinephrine or CL316,243, was
minimal compared to the results in A/J mice. It is interesting that the
inability of CL316,243 to prevent obesity in the high fat-fed B/6J mice
is coincident with the nearly complete absence of ß-adrenergic
responsiveness in IBAT of this strain. At this point we do not know
whether this lack of ßAR activity (particularly ß3AR
function) is responsible for the inability to respond to this compound.
However, preliminary data examining the effects of these diets with and
without CL316,243 indicate that within the first few weeks of
treatment, B/6J mice initially appear to respond to CL316,243, as
evidenced by significant decreases in the weights of selected fat pads
(our unpublished observations). Additional studies are in progress to
assess in greater detail the time course of body and adipose depot
weights, body composition, and expression and function of ßARs over
this chronic treatment regimen.
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displays representative Northern blots from A/J
mice, and Figs. 10 and 11 present
quantitative comparisons between A/J and B/6J mice. The results
indicate that excess adipose tissue mass due to high fat feeding is
associated with decreased expression of ß1AR and
ß3AR in WAT depots of both A/J and B/6J mice, whereas in
IBAT of high fat-fed mice, we actually observed a slight increase in
ß3AR messenger RNA (mRNA) levels. The paradoxical
decrease in ß3AR mRNA levels during treatment with
CL316,243 may be related to earlier studies by Granneman and colleagues
(30), in which they found that cold exposure (i.e. induction
of active thermogenesis) led to a decrease in ß3AR mRNA
in IBAT. Therefore, at least in IBAT, the relationship among
ß3AR transcripts, functional ß3ARs, and
thermogenic activity will require additional study.
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and 12, levels of UCP mRNA in IBAT were not significantly
reduced in either A/J or B/6J mice made obese by high fat feeding.
However, in the perirenal depot, which consists of a mixture of white
and brown adipocytes, there were significant decreases in UCP
expression in the high fat-fed, obese animals of both strains. Of
particular note, when the high fat diet was supplemented with the
ß3AR agonist, CL316,243, UCP mRNA levels were
significantly elevated in the interscapular depot of both A/J and B/6J
mice. Moreover, in perirenal adipose tissue of A/J mice, but not in
B/6J mice, UCP expression was completely restored. Most significantly,
the sc and gonadal white adipose depots from A/J mice consuming the
ß3AR agonist contained substantial quantitites of UCP
mRNA (Fig. 9). As there was little or no UCP in these depots in lean,
low fat-fed animals, these data suggest that the ability of CL316,243
to prevent obesity in A/J mice is related to increased thermogenic
activity in these peripheral depots as well as in the major
interscapular BAT depot. Consistent with this interpretation,
preliminary histological studies show that A/J mice treated with
CL316,243 displayed marked increases in brown adipocytes that express
immunoreactive UCP in several depots, including perirenal,
retroperitoneal, and gonadal (our unpublished observations).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have shown that B/6J mice raised on a high fat diet develop insulin resistance and diabetes (4). Moreover, analysis of white adipocyte depots indicates that high fat-fed, obese, B/6J mice display both adipocyte hyperplasia and hypertrophy. By contrast, A/J mice gain less body weight, do not develop diabetes, and show adipocyte hypertrophy without hyperplasia (5, 7). Our current results lend support to the idea that adipocyte adrenergic function is critical in the pathophysiology of diet-induced obesity and type II diabetes in the B/6J model. The ability of B/6J mice to dramatically increase their feed efficiency (weight gain/Cal consumed) is reminiscent of the "thrifty gene" hypothesis proposed by Neel 30 yr ago (34). Neel noted that ob/ob mice were able to survive food restriction much better than other strains. He went on to hypothesize that the genes responsible for diabetes were thrifty genes that evolved because of their adaptive value in an environment where food was scarce. Our results suggest that these genes reside in the B/6J background of the ob/ob mouse, and that they may be related to the ability of these animals to store fat when it is plentiful.
Regulation of adipose tissue metabolism has been shown to be impaired in genetically obese animals. Most studies have focused on the C57BL/6J obese (ob/ob) mouse, in which it has been shown that ß-adrenergic-stimulated lipolysis and cAMP production are significantly reduced (10, 11, 12). In earlier studies we reported that the expression and function of ß1AR and ß3AR are significantly depressed in adipose tissue of the ob/ob mouse (14). The results presented here on diet-induced obesity further underscore the importance of these earlier findings. Although the A/J and B/6J mice studied in the current work do not possess specific genetic mutations, the results show that certain genetic backgrounds combined with the consumption of a high fat diet can result in a similar impairment in ß-agonist-stimulated adenylyl cyclase activity (and decreased ßAR expression).
A finding of major significance in these studies is that the ß3AR agonist, CL316,243, could prevent diet-induced obesity and hyperinsulinemia in A/J mice. In marked contrast, chronic treatment with this ß3AR agonist did not prevent obesity or diabetes in B/6J mice. Our data also demonstrate that CL316,243 completely prevented the loss in adipocyte ßAR gene expression and function only in A/J mice. An equally revealing feature of these results is that only A/J mice showed substantial levels of UCP in such typical WAT depots as sc and gonadal fat. Thus, the effects of CL316,243 on weight may also be mediated by induction of brown adipocytes in WAT depots. This is consistent with observations of other investigators on the acute effects of CL316,243 treatment in rats (21). The decrease in ß3AR mRNA levels that we observed in IBAT from animals treated with CL316,243 may be related to earlier studies by Granneman and colleagues (30), in which they found that cold exposure (i.e. induction of active thermogenesis) led to a decrease in ß3AR mRNA in IBAT. However, the molecular basis for these results is not presently clear. As radioligands specific for ß3AR have not been developed, we do not know whether the level of ß3AR mRNA directly correlates with the amount of functional ß3AR protein in thermogenically active IBAT. For example, it is possible that upon stimulation of brown adipocytes, new ß3AR protein is synthesized in a process that leads to ß3AR mRNA degradation, similar to the translation-coupled degradation of c-myc mRNA (35, 36). This situation would result in a greater amount of functional ß3AR protein and less ß3AR mRNA. Another issue that should be addressed is whether the coupling of ß3AR in thermogenically active IBAT is more efficient, such that each mole of receptor protein is capable of generating a greater number of moles of cAMP. There are also several different adenyl cyclase isozymes that are expressed in brown adipocytes (17). Possibly, the expression of a particular form of adenylyl cyclase is increased during active recruitment of IBAT, again potentially leading to a greater turnover of ATP to cAMP per U ß3AR protein available. Experiments to examine these issues and other aspects of the regulation of the three ßARs in brown adipocytes should ultimately provide an explanation for our in vivo observations.
An important aspect of our experiments and one that distinguishes them from other studies of ß3AR agonists in mice is that the effect of CL316,243 to prevent obesity in A/J mice, but not B/6J mice, persists throughout a chronic treatment regimen. Thus, genetic background appears to be a key determining factor in both the propensity to develop obesity and the appearance in B/6J mice of clinical features of noninsulin-dependent diabetes mellitus. Although the most clearly identified function of BAT depots in rodents and human neonates is related to thermoregulation (37), the ability to pharmacologically prevent obesity and simultaneously recruit BAT in typically WAT depots supports the results of other studies, which indicated a critical role of BAT in the regulation of body composition (31, 32, 33).
In most human as well as rodent obesity, elevated levels of leptin are commonly found (38, 39, 40), and it has been suggested that a functional defect in the leptin receptor may exist, analogous to the diabetic (db/db) mutation in mice (41, 42). We have recently reported that chronic ß3AR agonist treatment can decrease leptin levels in diet-induced obese A/J mice (29), and that administration of recombinant leptin to ob/ob mice increases sympathetic outflow to IBAT (43). In view of our new findings reported here that high fat feeding precipitates a defect in adrenergic control of adipocyte function, particularly in B/6J mice, we propose that the high leptin levels frequently observed in obesity represent the result of a fat-induced disruption in the normal feedback that regulates adipocyte function. For example, in B/6J mice, in which this fat-induced defect is more severe than in A/J mice, diet-induced obesity is consequently more pronounced. Given the overwhelming evidence that most human obesity is linked to high fat consumption (2, 3, 9), it is likely that this mechanism plays an important role. Future studies that address the mechanism by which fat intake can lead to disruption of adipocyte adrenergic receptor function may, therefore, provide new insights into the pathophysiology of the most common forms of obesity.
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Acknowledgments |
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Footnotes |
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Received July 25, 1996.
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), a high fat diet (•), or a high fat diet
containing 0.001% CL316,243 (
). Animals were weighed at biweekly
intervals through the 16-week period on the diets. A, A/J mice; B, B/6J
mice.
