Departments of Medicine and Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425
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ABSTRACT |
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Obesity-resistant (A/J) and obesity-prone (C57BL/6J) mice were weaned onto low-fat (LF) or high-fat (HF) diets and studied after 2, 10, and 16 wk. Despite consuming the same amount of food, A/J mice on the HF diet deposited less carcass lipid and gained less weight than C57BL/6J mice over the course of the study. Leptin mRNA was increased in white adipose tissue (WAT) in both strains on the HF diet but to significantly higher levels in A/J compared with C57BL/6J mice. Uncoupling protein 1 (UCP1) and UCP2 mRNA were induced by the HF diet in brown adipose tissue (BAT) and WAT of A/J mice, respectively, but not in C57BL/6J mice. UCP1 mRNA was also significantly higher in retroperitoneal WAT of A/J compared with C57BL/6J mice. The ability of A/J mice to resist diet-induced obesity is associated with a strain-specific increase in leptin, UCP1, and UCP2 expression in adipose tissue. The findings indicate that the HF diet does not compromise leptin-dependent regulation of adipocyte gene expression in A/J mice and suggest that maintenance of leptin responsiveness confers resistance to diet-induced obesity.
uncoupling proteins; thermogenesis; adipose tissue
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INTRODUCTION |
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ACCUMULATION OF EXCESS ADIPOSE TISSUE is a known risk factor for hypertension, heart disease, and non-insulin-dependent diabetes mellitus. The epidemic of obesity in Western society is associated with consumption of high-fat (HF) diets. A subset of the obese population appears sensitive to the diabetogenic effects of HF diets in that their obesity progresses to insulin resistance and diabetes. A similar variation in sensitivity to dietary fat has been documented in mice (19, 43, 44, 54), where particular strains readily develop an obese/diabetic syndrome after chronic consumption of HF diets. These susceptible strains provide excellent models to study the developmental pathophysiology of an obesity syndrome that is highly analogous to human obesity (5, 44, 54). Contrasting fat-sensitive with fat-resistant mouse strains serves the dual purpose of illustrating the molecular adaptations used to avoid obesity and of identifying genes that are sensitive to dysregulation by HF diets. The common factor in mice that are resistant to dietary fat is an ability to avoid obesity despite increased caloric density, but the cellular adaptations that confer this resistance are poorly understood.
The ability of uncoupling proteins (UCP) to modulate energetic efficiency comes from their ability to short-circuit the mitochondrial proton gradient that drives ATP synthesis (30, 35, 39). A cardinal property of adipose tissue is that its thermogenic capacity is directly related to the amount of UCP expressed in it (31, 42). It is well established that leptin regulates expression of the UCP1 gene (4, 11, 31), whereas dietary factors may play an important role in controlling expression of UCP2 (1, 15, 47) and UCP3 (25, 51, 53) and regulating leptin responsiveness. Thus the ability of an animal to respond to increased caloric density may be dictated by its ability to increase thermogenic capacity. Using mouse strains that differ in their sensitivity to HF diets, we show that fat-resistant A/J mice, but not fat-sensitive C57BL/6J mice, increase thermogenic capacity in brown (BAT) and white adipose tissue (WAT) in response to HF diet. These differences may be the product of strain-specific changes in expression of and subsequent responses to circulating leptin.
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MATERIALS AND METHODS |
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Materials.
EDTA, sodium cholate, Triton X-100, BSA, guanidinium thiocyanate, TES,
sucrose, and other common chemicals were from Sigma Chemical (St.
Louis, MO). T1 RNase and TRIzol LS reagent were from Life Technologies
(Gaithersburg, MD); T7 RNA polymerase, SP6 RNA polymerase,
Taq DNA polymerase, Moloney murine leukemia virus reverse
transcriptase, and the pGEM-3Z cloning vector were from Promega
(Madison, WI); and the T7 Megashortscript kit was purchased from Ambion
(Austin, TX). 2-Mercaptoethanol was acquired from J. T. Baker
(Phillipsburg, NJ); oligonucleotide primers were prepared by the DNA
Core Facility at the Medical University of South Carolina;
Na[125I] and -[32P]cytidine triphosphate
were purchased from Du Pont NEN Radiochemicals (Boston, MA); and
Immobilon-P polyvinylidine fluoride membranes were from Millipore
(Bedford, MA). A semipurified HF diet was prepared by Research Diets
(New Brunswick, NJ) to contain 36% fat by weight (44),
and the low-fat (LF) control diet contained 5% fat, as described in
detail previously (44, 46). The fat source
for the diets was coconut and soybean oil (44). Male A/J
and C57BL/6J mice were obtained from Jackson Laboratories (Bar Harbor,
ME) at weaning. Serum insulin and leptin levels were estimated using
kits for rodent insulin and mouse leptin obtained from Linco Research
Labs (St. Charles, MO).
Experimental animal protocol. Fifty-six A/J and C57BL/6J mice were obtained at weaning, and eight animals of each strain were killed at time 0. One-half of the remaining animals of each strain were randomly assigned to receive either the LF or the HF diet (24 per strain per diet). The four groups of mice received their respective diets for 16 wk, and eight representative mice from each group were killed after 2, 10, or 16 wk on diet. At 2-wk intervals, food consumption was monitored in randomly selected groups of eight mice for 24-h periods. Animals were housed in a controlled environment at 22°C on a 12:12-h light-dark cycle with free access to food and water. Body weights were obtained twice weekly. Blood samples were obtained from each mouse at the time it was killed. Thereafter, interscapular BAT (IBAT), epididymal WAT (EWAT), and retroperitoneal WAT (RWAT) were carefully dissected from each animal for preparation of total RNA or isolation of adipocytes.
In an additional experiment, mice from each strain/diet combination were injected with vehicle or CL316,243 (1 µg· dayPreparation of RNA. After dissection, the interscapular, epididymal, and retroperitoneal fat pads were homogenized with TRIzol LS reagent using an Ultraturax (Tekmar, Cincinnati, OH) according to manufacturer's specifications. Total RNA was isolated and purified as previously described (21).
Ribonuclease protection assay.
RNA probes complementary to mRNA were produced by RT-PCR using total
RNA from mouse IBAT for UCP1 (5'-3':F, caatctgggcttaacgggt; R,
tgaaactccggctgagaag), UCP3 (5'-3':F, ccaccatggctgtgaagttcctg; R,
gggtgtacacctgcttgacggagtc), and 3-adrenergic receptor
(5'-3':F, accccagtgcagccaacacca; R, cgcaaccagtttcgcccaagg). Reverse
transcribed RNA from mouse EWAT was used for UCP2 (5'-3':F,
cagttctacaccaagggtc; R, aggtcataggtcaccagctca), and the UCP2 probe was
shortened to 143 bp by use of a Sma I digest, which cut the
fragment at a site corresponding to nucleotide 741. The respective
fragments were purified and cloned into the pGEM-3Z riboprobe vector
containing transcriptional start sites 5' and 3' to the multiple
cloning site (Promega, Madison, WI). The identities of the cloned
fragments were confirmed by sequencing, and the probes corresponded to
nucleotides 7-300 for UCP1, 741-884 for UCP2, 219-467
for UCP3, and 630-870 for the
3-adrenergic
receptor. The probe for mouse leptin was obtained from M. Daniel Lane.
The respective probes were labeled and used in our modification
(11, 21) of the ribonuclease protection assay
described by Granneman and Lahners (26). The protected
fragments were quantitated by comparison to known amounts of sense
strand RNA produced by SP6 transcription of the linearized plasmids and
incubated simultaneously with labeled probe. After digestion with 300 U
T1 RNase (Life Technologies), sense strand standards and protected
fragments were separated on 6% polyacrylamide/8 M urea gels and
visualized by autoradiography. Detected bands were quantitated by
scanning laser densitometry (Molecular Dynamics, Sunnyvale, CA). Bands
were standardized to the amount of 18S rRNA by cohybridizing with a
riboprobe complementary to the 18S rRNA (nucleotides 715-794).
Estimated concentrations of each mRNA were then determined by reverse
calibration from standard curves generated from the known amounts of
sense strand standards that were included in each assay.
Adenylylcyclase assay on adipocyte plasma membranes.
Adipocytes were isolated from the epididymal fat pads and used to
prepare plasma membranes, as described in detail previously (21, 22). The plasma membranes were used in
adenylylcyclase assays to assess the functional coupling of the
3-adrenergic receptor to its effector system among the
treatment groups, as described previously (8,
20, 21).
Methods of analysis.
The estimated concentrations of UCP1, UCP2, UCP3, leptin, and
3-adrenergic receptor mRNA were obtained by reverse
calibration from standard curves, as described in Ribonuclease
protection assay. Group means for mRNA estimates, growth data, and
plasma hormone concentrations were analyzed by means of a three-way
factorial design with strain, diet, and time as main effects. The
strain × diet × time interaction was tested by residual
variance (animal within strain × diet × month) as the error
term, and in the absence of such interactions, interest shifted to
strain × diet differences within each time point. Post hoc
testing of group means within each time point was made with the
Bonferroni correction, which used the pooled error term to calculate
standard errors. Protection against Type I errors was set at
5% (
= 0.05).
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RESULTS |
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Growth and food consumption.
Body weights of mice within each strain were similar at the beginning
of the study (Fig. 1), as were the
weights of epididymal fat pads from representative mice of each strain
(Table 1). Fat pad weights were also
similar between the strains after mice consumed the LF diet for 2, 10, and 16 wk (Table 1). In mice mice consumed the LF diet, body weights
began to diverge after 4-5 wk, such that the C57BL/6J mice were
heavier (P < 0.05) at all subsequent time points (Fig.
1). The response between mouse strains on the HF diet was more
pronounced, and Fig. 1 illustrates that the C57BL/6J mice grew faster
and to higher weights than the A/Js (P < 0.05). The
strain difference was evident as early as 2 wk, and the fat pad weights
in the C57BL/6J mice were higher (P < 0.05) than those of the A/Js at the latter two time points (Table 1).
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WAT gene expression.
To examine the basis for the apparent differences in plasma leptin
between A/J and C57BL/6J mice, we examined leptin mRNA in WAT from both
strains. Leptin mRNA levels in EWAT were comparable between strains at
the beginning of the study and changed little over time in either group
consuming the LF diet (Table 2). In contrast, differences were clearly evident in mice consuming the HF
diet, because leptin mRNA levels were significantly higher (P < 0.05) in A/J compared with C57BL/6J mice at the
2-wk time point (Table 2). This difference was also evident at 10 and
16 wk, because leptin mRNA levels were 50-100% higher in A/J than in C57BL/6J mice on the HF diet (Table 2). Leptin mRNA levels were also
higher (P < 0.05) in RWAT of A/J compared with
C57BL/6J mice (0.043 ± 0.006 vs. 0.020 ± 0.006 fmol
mRNA/µg RNA) consuming the HF diet at the 10-wk time point. Using two
different depot sites, we confirmed the results that leptin expression
is unexpectedly higher in A/J compared with C57BL/6J mice consuming the
HF diet.
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BAT gene expression.
BAT UCP1 mRNA levels were similar between C57BL/6J and A/J mice
(4.22 ± 0.22 and 3.74 ± 0.12 fmol UCP1 mRNA/µg RNA) at
the start of the experiment and between A/J and C57BL/6J mice on the LF
diet at all but the initial time point over the course of the study
(Fig. 6; Table 2). UCP1 mRNA levels were
also similar between the mouse strains after 2 wk on the HF diet, but
at both 10 and 16 wk, UCP1 mRNA was significantly higher
(P < 0.05) in BAT from A/J compared with C57BL/6J mice
(Fig. 6; Table 2). Thus A/J mice responded to the HF diet by increasing
UCP1 mRNA levels, whereas the C57BL/6J mice failed to respond in a
comparable manner.
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DISCUSSION |
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The translated product of the ob gene regulates food consumption by communicating the status of peripheral adipose stores to the brain (17, 55). Recent evidence indicates that leptin also regulates energy expenditure through the sympathetic nervous system by enhancing UCP1 expression in both BAT and WAT (2, 10, 11, 32, 33). The consensus is that leptin is the afferent signal in a feedback loop between adipose tissue and the brain that acts to regulate both energy intake and expenditure. This paradigm predicts that animals will respond to diets of high caloric density by reducing food intake and increasing energy expenditure. In the present work, C57BL6/J mice failed to fulfill this prediction and are among several strains of mice classified as "sensitive" to HF diets (5, 43, 44). In contrast, A/J mice are classified as "fat resistant," because they do not develop the associated diabetic pathologies on HF diets and become only moderately obese (43, 44). The present findings are consistent with these classifications and show that C57BL/6J mice grow faster and deposit significantly more fat than A/J mice at similar or lower rates of food consumption. Taken together, these findings demonstrate a fundamental difference in energy requirements for body weight maintenance between these mouse strains and argue that the ability of leptin to match rates of food intake and energy utilization was differentially compromised by the HF diet. Therefore, the major goal of the present study was to examine the regulation of leptin expression and function as a basis for the differing propensities of C57BL/6J and A/J mice to become obese.
Plasma leptin levels were initially similar between the mouse strains, but after 2 wk on the HF diet, leptin levels were higher in A/J than in C57BL/6J mice. The data suggest that plasma leptin was also higher in A/J mice at 10 and 16 wk, but the variability of the observations precluded detection of this difference. Surwit et al. (45) reported a similar disproportionate increase in plasma leptin in A/J compared with C57BL/6J mice after 2-4 wk on HF diet. This is surprising, given the greater fat deposition in C57BL/6J compared with A/J mice, and it suggests that leptin expression per unit of adipose tissue was actually higher in A/J compared with C57BL/6J mice. To test this hypothesis, we compared leptin mRNA levels in two WAT depot sites from mice of each strain. The findings from these studies are consistent with our hypothesis at all time points. These differences are particularly important because they occurred during the initial 10 wk of the study, when fat deposition was clearly occurring at a faster rate in C57BL/6J compared with A/J mice. Given its demonstrated role in regulating energy utilization and fat oxidation (10, 11, 32, 33, 52), the higher leptin expression in A/J compared with C57BL/6J mice may have contributed to the observed differences in fat accumulation by increasing peripheral energy utilization in A/J mice. Support for this suggestion comes from Surwit et al., who reported that core temperatures were higher in A/J vs. C57BL/6J mice consuming HF diets. Lower body temperature is a distinguishing characteristic of ob/ob mice, and replacement of the missing leptin restores body temperature to normal (40), ostensibly by increasing UCP1 expression (11) and thermogenic activity in BAT. Similar observations were evident in the present study, where the higher leptin expression in A/J mice on HF diets was mirrored by higher UCP1 mRNA levels in BAT from these mice. These findings support the concept that disproportionately higher leptin expression in A/J compared with C57BL/6J mice lessens their energetic efficiency by increasing nonshivering thermogenesis.
The hypothesis that high caloric density mobilizes a more vigorous thermogenic response in A/J compared with C57BL/6J mice is also supported by measurements of UCP2 mRNA, where levels were significantly higher in WAT from A/J mice. These findings are similar to results reported by Fleury et al. (15) and Surwit et al. (47), with two subtle but important differences. First, our results show that UCP2 mRNA expression in adipose tissue was comparable between the strains at weaning, with strain and diet-associated differences developing thereafter. Second, the present work shows that UCP2 mRNA levels were consistently higher in A/J than in C57BL/6J mice after 2, 10, and 16 wk on the HF diet. The previous study (15) had found a similar difference at early time points but actually found higher levels of UCP2 mRNA in C57BL/6J compared with A/J mice later in that study. Although UCP2 has been shown to lower yeast membrane potential and uncouple respiration (15, 34), the functional significance of increased UCP2 mRNA in the present system is unknown. If increased mRNA levels are matched by increased protein expression, then it seems likely that decreased metabolic efficiency would result. In contrast to UCP1 and UCP2, no evidence was found to support a role for UCP3 in diet-induced obesity.
The mechanism for increased UCP2 expression in A/J compared with
C57BL/6J mice is unknown. Although initial reports suggested that UCP2
was regulated by leptin (56) or by norepinephrine (3), a number of other studies have questioned these
findings (11, 15, 24,
25). For instance, we showed that UCP2 was unchanged in
retroperitoneal WAT from mice in which leptin treatment induced UCP1
expression in the same tissue (11). Therefore, it seems
unlikely that the higher leptin expression in A/J mice is the basis for
the observed strain differences in UCP2 mRNA. What seems more likely is
that UCP2 expression was differentially affected between strains by
dietary fat. Support for this suggestion comes from reports showing
that UCP2 expression is responsive to changes in circulating free fatty
acids (25, 41), and from the work of Aubert
et al. (1), who showed that peroxisome
proliferator-activated receptor-2 (PPAR
2)
agonists increased UCP2 mRNA in adipocytes. Given that fatty acids can
transcriptionally activate PPAR
-sensitive genes (16)
and the recent report showing that PPAR
expression level influenced
adipocyte gene expression (36), the possibility of strain
differences in signaling through this pathway warrants further study.
Observed differences in circulating leptin and tissue mRNA suggest that
regulation of this gene differs between the two mouse strains. Based on
recent reports showing that leptin expression is inhibited by
adrenergic stimulation of adipose tissue (9, 10, 48, 49), we explored the
possibility that inhibitory regulation of leptin expression was
differentially compromised in mice of each strain by the HF diet. The
results from studies using a selective 3-adrenergic
receptor agonist were surprising and showed that the HF diet
completely transformed the inhibitory response in each mouse strain.
For instance, CL316, 243 failed to reduce leptin mRNA in A/J mice
consuming the LF diet, whereas A/J mice on the HF diet showed a robust
agonist-induced reduction in leptin mRNA. In contrast, the
CL316,243-mediated reduction in leptin mRNA noted in C57BL/6J mice on
the LF diet was essentially absent in C57BL/6J mice on the HF diet. A
potential explanation for diet-induced transformation of the response
could be changes in
-adrenergic receptor expression. Collins et al.
(7) examined this question in C57BL/6J and A/J mice fed HF
diets for 16 wk and found greater reductions in
3-adrenergic receptor mRNA and function in WAT from
C57BL/6J mice. We found comparable reductions in
3-adrenergic receptor mRNA and function in C57BL/6J mice
at 16 wk, but the observed differences in inhibitory regulation of leptin expression occurred at earlier time points (2 and 10 wk), when
we found no evidence of compromised expression or function of the
receptor. Although leptin expression involves a number of hormonal
inputs (12-14, 38), a recent study
showed that reduced expression of PPAR
in adipose tissue had a
profound effect on leptin expession levels (36). Leptin
expression was disproportionately high in relation to adipose tissue
mass, producing leaner mice with elevated metabolic rates
(36). In that study, the authors showed that changes in
regulatory factors and systems that impact adipocyte gene expression
are capable of transforming obesity-prone mice into mice that are
resistant to diet-induced obesity (36).
It is reasonable to assume that differences in inhibitory regulation of leptin expression are not the sole basis for the differences noted in the present work. Our results, however, make a strong case that leptin expression and its regulation are fundamentally different in WAT from A/J and C57BL/6J mice. Because cAMP-dependent mechanisms are also important in regulating leptin release from adipocytes (18), it will be important to establish whether the observed strain differences in gene transcription extend to leptin release from the adipocyte.
Strain and diet-dependent differences in adipocyte gene expression could also be related to differences in the proliferative state within adipocyte depots. For instance, diet-induced differences in hyperplastic growth within white fat depot sites could impact circulating leptin levels through an increase in leptin expressing cells. Although cell numbers were not determined in the present study, Surwit et al. (43) found a 40% increase in adipocytes in white fat depots from C57BL/6J mice that had consumed the high-fat diet for 16 wk. In contrast, no evidence of hyperplasia was found in A/J mice on the same diet (43). Therefore, these findings are inconsistent with the suggestion that increased leptin expression in A/J mice is caused by adipocyte hyperplasia; rather, these data support the conclusion that leptin expression per adipocyte is higher in WAT from A/J compared with C57BL/6J mice.
Recent evidence suggests that diet-induced obesity is associated with
the development of leptin resistance (28, 37,
50). Our studies show that, despite robust increases in
circulating leptin, strain differences in body weight and fat accretion
occurred at similar rates of food consumption. These findings suggest
that strain differences in energy utilization reflect either a relative leptin insufficiency or a compromised ability of leptin to induce thermogenic capacity and activity in C57BL6/J compared with A/J mice.
The present studies do not distinguish between these possibilities, but
it should be noted that several mouse models of diet-induced obesity
display peripheral but not central leptin resistance (6, 50). Leptin resistance may also occur through diet-induced
effects on signaling components in target tissues. An example would be the diet-induced reduction of 3-adrenergic receptor
expression in C57BL/6J mice, which could limit the ability of adipose
tissue to respond to sympathetic stimulation. This seems unlikely in the present study, because differential fat accumulation occurred well
before any change in
-adrenergic signaling efficacy. Our findings
are more consistent with changes in downstream signaling components
that modify the set points for leptin release between the two mouse
strains. The present studies do not exclude central leptin resistance
as a contributing mechanism of diet-induced obesity; rather they
demonstrate fundamental differences in the regulation of adipocyte gene
expression between A/J and C57BL/6J mice. It will be important in
future studies to assess the relative importance of altered leptin
expression vs. central and peripheral mechanisms of leptin resistance
in various models of diet-induced obesity.
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ACKNOWLEDGEMENTS |
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The authors acknowledge the excellent technical assistance of Isabel Frampton and Jami Kelley.
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FOOTNOTES |
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This work was supported by a research grant from the American Diabetes Association (T. W. Gettys), National Institute of Diabetes and Digestive Kidney Diseases Grant DK-53981 (T. W. Gettys), and Research Grant No. 9800699 from the US Department of Agriculture NRICGP/USDA (T. W. Gettys).
Address for reprint requests and other correspondence: T. W. Gettys, 916-G Clinical Science Bldg., Medical University of South Carolina, 96 Jonathan Lucas St. Charleston, SC 29425 (E-mail: gettystw{at}musc.edu).
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.
Received 15 December 1999; accepted in final form 8 March 2000.
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