Department of Neuroscience, Amgen, Thousand Oaks, California 91320
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ABSTRACT |
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The role of estradiol in mediating leptin's effects on body weight was assessed in ovariectomized (OVX) mice before and after the onset of obesity. Ovariectomy did not alter leptin levels before the onset of obesity, and estradiol adminstration (0.05-17 µg/day for 14 days) did not significantly alter leptin levels if they were corrected for the estradiol-induced reduction in body fat. The converse was also true, in that leptin administration (0.4-140 µg/day) did not alter estradiol levels in intact mice. Furthermore, neither estradiol reduction (via ovariectomy) nor addition (via exogenous administration) significantly altered leptin's ability to reduce fat mass. Leptin was equally effective in reducing body weight in lean or obese OVX mice and intact controls. Finally, estradiol did not change the magnitude of leptin's effect on fat mass reduction when it was given in combination with leptin to lean intact or OVX mice. Estradiol may have indirectly affected leptin efficacy, because leptin did not produce as large a change in fat mass at lower doses in lean OVX mice as it did in intact counterparts. Taken together, these data suggested that 1) estradiol does not directly regulate leptin secretion or its effects on fat mass and 2) leptin does not directly regulate estradiol secretion or its effects on fat mass. Leptin and estradiol, however, may interact in an indirect fashion to affect fat utilization.
leptin; estradiol; ovariectomy; efficacy; body fat
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INTRODUCTION |
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LEPTIN is a 16-kDa protein that is secreted from adipose tissue in a pulsatile fashion (26, 42, 49). Exogenous administration of leptin results in weight loss, reduced adiposity, and appetite suppression (3, 14, 35). Although leptin level correlates well with body weight and adiposity (6, 9, 15, 22, 27, 29), the association between leptin level and body weight is not the same in both genders. Several groups have found that females have more leptin than males (19, 21, 34, 38, 39), even when 1) both groups are normalized for body fat (19, 21, 34, 38, 39); 2) lean males and females are compared (19); 3) children are used as subjects (17); or 4) venous cord blood samples from newborn males and females are compared (30).
One explanation for the gender difference in leptin levels is that ovarian hormones modulate leptin secretion and, hence, energy balance. The idea that ovarian hormone regulation could be linked to energy balance is an old one. Food deprivation, glucoprivation, and lipoprivation all can affect estrogen receptor number, estrus cycling, and lordosis (46). These manipulations also have negative effects on puberty onset and human fertility (10). Interestingly, leptin can counteract the effects of food deprivation on puberty onset in both males and females (1, 4). Furthermore, leptin levels are reduced in human females with amenorrhea due to anorexia (26). There is also evidence that the leptin receptor is localized on the ovary (5, 48). Finally, estradiol itself can have effects on food intake and body weight that are in the same direction as leptin, in that it causes a transient reduction in food intake and a moderate reduction in body weight in ovariectomized females (2, 33, 44, 46).
It is possible, then, that estrogen could be involved in the mechanism by which leptin regulates adiposity in females. Furthermore, if estradiol and leptin have similar effects on body mass and adiposity, it is possible that estradiol given in combination with leptin, or given as a pretreatment before leptin administration, could act to enhance the effects of leptin on adiposity in females. To test these hypotheses, we 1) assessed the effects of estradiol addition (continuous subcutaneous administration) or estradiol subtraction (ovariectomy) on leptin levels, 2) assessed leptin's ability to reduce body weight and fat mass in lean or obese ovariectomized (OVX) and intact mice, and 3) evaluated the effects of either simultaneous administration or pretreatment with estradiol on leptin's ability to reduce fat mass in lean and obese OVX mice.
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METHODS |
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Experiment 1 (lean OVX mice).
The importance of the ovary in mediating leptin's effects on body
weight was tested in three ways. First, the effects of estradiol elevation were assessed by measuring serum leptin in 36 intact mice
treated with 17-estradiol pellets that delivered either 0.0, 0.05, 0.5, 1.2, 2.4, or 17 µg/day for a period of 2 wk. The 0.0 (control)
pellet contained the solid vehicle only. Second, the impact of ovarian
hormone level reduction was tested by measuring leptin levels in
PBS-treated OVX mice 4 wk after surgery. Third, the effects of
ovariectomy on leptin efficacy were tested by comparing leptin's
metabolic effects on OVX vs. intact mice. The comparison was done
before the weight gain that eventually occurs in OVX rodents (2, 31,
33). Forty-two OVX and 42 sham-operated C57Bl6J mice (SHAM) (9 wk of
age) were obtained from Charles River Laboratories. OVX and SHAM mice
were subdivided into seven dose groups, with vehicle controls for both
groups. Two weeks after surgery, 1)
recombinant methionine murine leptin (r-MetMuLeptin; 0.03-12.6
mg/ml, or 0.4-140 µg/day), 2)
a combination of 17
-estradiol (17 µg/day sc pellets) and leptin
(120 µg/day), or 3) PBS was infused subcutaneously by use of an osmotic minipump (Alzet 1007D) that
delivered 0.5 µl/h. Leptin, the leptin-estradiol combination, or PBS
was administered for 14 days.
Experiment 2 (obese OVX mice). In the second experiment, 24 OVX mice were allowed to gain weight and were used as an alternative model of obesity in which to test leptin efficacy after a 4-wk pretreatment with estradiol. Estradiol (EST) or placebo (CON) treatment (17 µg/day sc pellet) began 15.5 wk after OVX or sham surgeries. Four weeks after the onset of hormone therapy, EST and CON groups were further subdivided into either PBS or leptin groups. r-MetMuLeptin (10 mg/ml) or PBS was administered via continuous subcutaneous infusion (Alza osmotic mini-pumps, 0.5 µl/h, 2001D) for 14 days. Body weight was measured daily. Blood was collected 2 days before the onset of leptin or PBS treatment and on the last day of leptin or PBS infusion. Mice were euthanized on the last day of leptin treatment, and carcasses were taken for composition analysis.
Mice from all experimental groups were individually housed in the Amgen vivarium, with the ambient temperature set at 21-23°C and a 12:12-h light-dark cycle in effect (6:30 AM on; 6:30 PM off). All mice were allowed both food and water ad libitum. The procedures described above were approved by the Laboratory Animal Research Committee at Amgen.Carcass composition analysis. Carcass composition was assessed with the methods of Leshner et al. (24). Water composition was determined by subtraction of carcass weight before and after a 5-day dehydration period. Fat was extracted with ethyl ether and ethyl alcohol from a preweighed portion of the ground, dried carcass, so that percent fat could be calculated from the amount of material remaining after the extraction procedure. Lean mass was defined as the proportion of ground carcass that remained after dehydration and ether extraction.
Serum chemistry.
Serum nonesterified free fatty acids (NEFA), cholesterol,
-hydroxybutyric acid (BHBA), and triglycerides were analyzed using a
Hitachi 717 blood chemistry analyzer. Corticosterone was measured using
a competitive immunoassay system developed by Boehringer Mannheim
Biochemicals (Indianapolis, IN). Leptin levels were measured using a
solid phase sandwich enzyme immunoassay, with affinity-purified polyvalent antibody immobilized in microliter wells. Leptin was calculated from standard curves generated for each assay by use of
recombinant mouse leptin. The detection limits of the assay were 70 pg/ml (38). Estradiol was measured using RIA technology on serum that
was extracted on Sephadex LH20 columns by following the methods
described by Goodman (12) and Resko et al. (37).
Statistics. Repeated-measures ANOVA was used for all comparisons between intact and OVX groups over time, with least squares analysis used as the post hoc test for dissecting interaction terms. In the case of single time-point data, 1-, 2-, or 3-way ANOVA was chosen as the analysis method for main effects, with Fisher's least significant difference test used as the post hoc test. Simple regression was used to correlate leptin levels with carcass fat in estradiol-treated mice, and half-maximal effective dose curves were fitted using a third-order sigmoid equation.
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RESULTS |
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Experiment 1 (lean OVX mice).
Estradiol administration reduced serum leptin in a dose-dependent
manner [F(5,29) = 8.01;
P < 0.0001] (Fig.
1A),
but the reduction correlated significantly with the effects of
estradiol on carcass fat (r = 0.84;
P < 0.0001), which can be observed
in Fig. 1B. Therefore, if leptin level
was corrected for carcass fat, there was no longer a significant effect
of estradiol on leptin level [F(5,29) = 2.2;
P < 0.07]. Ovariectomy, which
reduced estradiol levels to approximately one-third of intact levels,
did not significantly alter leptin levels 4 wk after surgery
[F(1,18) = 0.42;
P < 0.52], as is shown in Fig.
1C.
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Experiment 2 (obese OVX mice).
Nineteen to twenty weeks after surgery, OVX mice weighed significantly
more (15.3%) than intact controls
[F(1,34) = 29.8; P < 0.01 (post hoc
P < 0.0001)]. Leptin levels in
these OVX mice were also significantly higher (4-fold) than in controls
at the above time point [F(1,33) = 26; P < 0.0001 (post hoc
P < 0.0001)] (Table
3). After 4 wk of estradiol pretreatment at
a high dose (17 µg/day), OVX mice weighed significantly less than
placebo-treated counterparts (P < 0.0008), which was reflected by a significant surgery × hormone
interaction [F(1,34) = 22.5;
P < 0.0001]. In contrast,
estradiol-treated intact mice weighed more than placebo controls
(P < 0.004). Leptin levels were
significantly lower in estradiol-treated mice, regardless of surgical
treatment, as is shown by a significant hormone effect
[F(1,33) = 47.9;
P < 0.0001]. As in
experiment 1, leptin levels in
estradiol vs. PBS-treated mice correlated with body fat in both OVX and
intact mice (r = 0.89;
P < 0.0001). All of these effects
are shown in Table 3 in columns concerned with baseline weight and
baseline leptin levels.
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DISCUSSION |
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Exogenous estradiol did not significantly affect serum leptin if the data were corrected for the fat loss induced by estradiol. Estradiol's lipolytic effects and the association of leptin level with fat mass are both well-documented phenomena (6, 7-9, 15, 22, 27, 29, 45). Therefore, it is unlikely that estradiol directly affects leptin secretion. Consistent with this, lean OVX mice had leptin levels that were similar to intact controls, suggesting that a reduction in estradiol also did not alter leptin levels. Our ovariectomy data, however, are not in agreement with data showing a reduction in leptin levels (41) and ob gene expression (41, 47) in rats 2-8 wk after surgery, despite the fact that fat mass is typically increased during this time period in rats (31). The reason for the inconsistency is unclear. There is some disagreement in the human subject literature, as well, regarding the relationship between estradiol level and leptin level. Havel et al. (18), for example, showed that leptin levels were not different in pre- and postmenopausal women if the levels were matched for fat mass, and that hormone replacement therapy did not alter leptin levels in postmenopausal women. Shimizu et al. (41) and Rosenbaum et al. (38), however, concluded by use of correlational methods that leptin levels were reduced in postmenopausal women. Finally, Messinis et al. (32) reported that leptin levels increased along with estradiol levels during the second half of the follicular phase of the menstrual cycle. Therefore, the relationship between estradiol and leptin levels appears to be a complex one, which may be dependent on factors such as the subjects' metabolic status and age.
The finding that exogenous leptin administration induced similar weight loss in OVX and intact mice further supports the hypothesis that estradiol does not directly regulate leptin production or functional activity. In addition, combined leptin and estradiol treatment did not reliably reduce fat mass more than leptin alone at a dose that was much lower than that used in the combination treatment. Again, the above evidence suggests that the two hormones do not act directly through a shared receptor or signaling pathway. Taken together, these data suggest that neither the ovary nor estradiol or estradiol receptors (by inference) are required for leptin-induced weight loss.
Ovarian hormones may induce a metabolic state that influences leptin's effects on fat mass, however, because leptin-induced fat loss was significantly attenuated in OVX mice compared with intact counterparts. For example, ovariectomy could have caused metabolic or neuroendocrine changes that indirectly affected leptin-induced fat loss before obvious weight gain. Cholesterol, triglycerides, BHBA, and corticosterone were indeed altered by ovariectomy, in that cholesterol and corticosterone were increased, whereas triglycerides and BHBA were reduced. All of these serum chemistry changes are consistent with previous observations in OVX animals (11, 20, 28).
Whether the observed increase in cholesterol levels or reduction in triglycerides would affect leptin's ability to reduce fat mass is unclear. Leptin has been shown to reduce carcass fat in animals with elevated cholesterol (16, 25, 36), but there are no data that address the effects of leptin in animals with reduced serum triglycerides, as observed in OVX mice.
It does not appear that OVX mice are incapable of responding to the effects of leptin on fat mobilization, because serum NEFA level was significantly increased in OVX mice, even at very low doses of leptin. Perhaps the intracellular FFA oxidation response to stress or energy deprivation is somehow impaired in OVX animals. In view of several reports suggesting that leptin reduces carcass fat by increasing intracellular FFA oxidation (40, 50), it is possible that leptin's effects on FFA oxidation could be blunted in an organism with an impaired FFA oxidation response to stress or fuel deprivation.
Leptin administration also reduced fat mass and body weight in OVX mice that were allowed to become obese. Nineteen to twenty weeks after surgery, OVX mice weighed 15% more than intact (sham) controls, an observation that is in agreement with earlier reports (2, 31, 43). These mice had 3-4 times more carcass fat and 3-4 times higher leptin levels than intact controls, which is consistent with the idea that leptin correlates very closely with body fat (6, 9, 15, 22, 27, 29). Two weeks of continuously infused leptin reduced body fat by ~40% in OVX mice. Fat mass appeared to be reduced to a greater degree in intact counterparts, in that it was reduced to undetectable levels. Again, this could suggest that leptin and estradiol may interact in an indirect fashion, with both hormones eventually affecting fat utilization through a common final pathway (as was suggested for the lean OVX mice). Such an interpretation is complicated for this obese set of OVX mice by the fact that animals with increased fat mass do not respond to low-dose leptin with the same magnitude of fat loss as do lean counterparts (3, 13, 36), which is why it was important to assess leptin efficacy in OVX mice that had not yet become obese.
Leptin was as effective in reducing total body weight in OVX mice as it was in intact controls, as demonstrated by similar net weight loss after 14 days of treatment for the two groups. Leptin, then, reduced total body weight more effectively in OVX-induced obesity than reported in diet-induced obese AKR mice or in obese female CD1 mice, where lower doses of leptin did not produce as great a magnitude of weight loss as in lean controls (3, 13, 36). It should be emphasized, however, that our OVX mice were not as "obese" as the diet-induced AKR mice or the obese female CD1 mice (in terms of body weight) that were used as subjects in the cited studies, and low-dose leptin causes a greater reduction in body weight in lean animals than in obese counterparts (13, 36).
Four weeks of estradiol treatment also reduced body weight in obese OVX mice, a result that is in agreement with previous work (2, 43, 45). After the 4-wk treatment period, OVX mice treated with estradiol were only slightly heavier than untreated, intact controls, and they and intact mice had very similar levels of fat and serum leptin. Subsequent leptin treatment along with continued estradiol, however, reduced all of these variables even further, to the point where body weight was lower than in intact mice or OVX mice treated with estradiol alone, and carcass fat was reduced to undetectable levels, as it was in intact mice treated with leptin. This result is not particularly surprising, because estradiol pretreatment reduced body fat and leptin levels, rendering a "lean" phenotype to these OVX mice, as can be inferred from the comparison of baseline weight for OVX/EST/PBS groups with that of either SHAM/CON/PBS or SHAM/EST/PBS groups in Table 3. As we have mentioned, lean animals are more sensitive than obese counterparts to leptin's effects on body weight at low to moderate dose ranges (13, 36). This, along with the fact that fat loss was not increased by simultaneous administration of leptin and estradiol in lean OVX mice, suggests that leptin was not additive to estradiol pretreatment in obese OVX mice.
A similar pattern of effects on fat mass were observed in intact mice pretreated with estradiol and given leptin, although total body weight was actually increased with estradiol adminstration in intact mice and was normalized with subsequent leptin treatment. The effect of estradiol on body weight in intact mice was probably due to its effects on carcass water, because water was also increased by chronic estradiol treatment and normalized by leptin.
All of the above addresses whether estradiol could either directly or indirectly affect leptin secretion or function. Some of our data also address the converse: that is, whether leptin acts by affecting estradiol production. The data in this study demonstrated that leptin administration did not significantly affect serum estradiol levels, which is in agreement with data showing that leptin did not alter basal granulosa cell number or production of estradiol in vitro (43, 48), despite the fact that leptin receptors have been localized on ovary tissue (5). Perhaps these receptors act to mediate leptin's inhibitory effects on estradiol production in states of metabolic stress, as would be the case when insulin is elevated (43).
Taken together, our results suggest that leptin does not directly affect estradiol production, because leptin administration does not alter estradiol levels. In addition, estradiol does not directly affect leptin production or its effects on fat mass reduction, because 1) exogenous estradiol does not alter leptin levels independently of its effects on fat mass, 2) the effects of leptin on body weight are the same in OVX and intact mice, and 3) the effects of combined estradiol and leptin treatment on fat mass are not consistently different from the effects of leptin alone. In addition, the ovary is not a necessary component of leptin's effects on metabolism, because OVX mice show leptin-induced weight loss and fat reduction, whether the mice are lean or obese. Ovarian hormones, however, may have indirect effects on these variables, since both lean and obese OVX mice show a moderate attenuation of leptin-induced fat loss. Therefore, whereas estradiol may indirectly affect leptin efficacy or secretion, it is unlikely that estradiol acts directly on leptin receptors or its signaling pathway to reduce fat mass.
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ACKNOWLEDGEMENTS |
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We are indebted to Larry Ross (Amgen Pathology Department) for measurement of the serum chemistry variables, to Margery Nicolson and Jason Moore (Amgen Neuroscience) for measurement of serum leptin, and to David Hess (Oregon Regional Primate Center) for measurement of serum estradiol.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for correspondence and reprint requests: M. A. Pelleymounter, Dept. of Neuroscience, Neurocrine Biosciences, 10555 Science Center Dr., San Diego, CA 92130 (E-mail: Mpelleymounter{at}neurocrine.com).
Received 16 November 1998; accepted in final form 4 February 1999.
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