Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, New York 12208
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ABSTRACT |
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Midlife weight gain occurs in many species, suggesting that leptin signaling is impaired at middle age. To test this hypothesis, we measured changes in food intake and body composition in young (Y) and middle-aged (MA) C57BL/6 male mice infused subcutaneously with phosphate-buffered saline or leptin. Leptin-induced decreases in food intake and body fat were delayed in MA mice and associated with catabolism after longer treatment periods. Endogenous plasma leptin levels did not correlate with body fat in MA mice. Calorie restriction (CR) reduced body fat, plasma leptin, and insulin in MA mice to levels in Y mice but did not upregulate leptin sensitivity. CR mice did not respond to leptin doses that inhibited food intake in MA mice and reduced food intake and body fat in Y mice significantly below levels in CR mice. Plasma corticosterone was significantly higher in leptin-treated CR vs. MA mice. We conclude that MA C57BL/6 mice exhibit impaired leptin signaling and that CR, possibly by elevating glucocorticoids, impairs appetite control without improving the metabolic actions of leptin.
weight gain; food intake; glucocorticoids; insulin; longevity
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INTRODUCTION |
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WEIGHT GAIN AT MIDDLE AGE occurs in at least 50% of the population and has been linked to diabetes, cardiovascular disease, stroke, and reduced lifespan. Conversely, avoiding midlife weight gain, whether by virtue of genes, diet, or exercise, increases longevity (4, 10, 24). Understanding the etiology of this increase in body weight will aid in improving the length and quality of life.
Weight gain in adulthood is almost exclusively in the form of fat (4). Body fat levels are signaled by the adipocyte hormone leptin, which is normally produced in proportion to fat mass and acts on the brain to decrease appetite and increase metabolic rate (11). Greater adiposity in middle age could be caused by decreased leptin sensitivity, such that slower or smaller leptin-induced changes in food intake and metabolism fail to prevent further fat deposition. Indeed, leptin has been shown to inhibit food intake and stimulate energy expenditure less effectively in aged (24-mo-old) rats (15, 21, 22, 25). Weight gain can also occur if leptin production decreases during aging, thus decreasing inhibitory control of food intake and fat accretion. This possibility has been suggested by the lack of positive correlation between plasma leptin and body fat in older humans (16). However, because these studies either used relatively aged subjects (15, 21, 22, 25), or pooled middle-aged with old individuals (16), it is not clear whether decreases in leptin responses or levels occur specifically by middle age, when weight gain is most relevant to life expectancy. To test the hypothesis that deficits in leptin signaling occur by middle age, we have compared effects of leptin infusion on food intake and body composition in 2- and 12-mo-old male C57BL/6 mice. These ages correspond respectively to young maturity and the phase of postmaturity weight gain in this strain (27).
Calorie restriction that minimizes age-associated weight gain has been shown to extend lifespan in experimental rodents and primates (7, 14). Many of the benefits of reduced food intake and body weight are attributed to improvements in insulin sensitivity (4). However, the high failure rate of dieting in the general population suggests that lifelong calorie restriction is unlikely to be a successful approach to improving public health. Because many of the effects of food deprivation are attributed to decreased leptin levels (2), leptin treatment could facilitate dietary restraint by promoting the weight-reducing, while mitigating the appetite-stimulating, effects of calorie restriction. We hypothesized that middle-aged, chronically calorie-restricted (CR) mice would be more sensitive to leptin than their age-matched, free-feeding counterparts, in part because lower endogenous leptin levels might upregulate recalcitrant signaling mechanisms. In addition, because we have found that elevated insulin can be associated with leptin resistance (26), we also hypothesized that lower insulin levels in CR mice would be associated with improved leptin responsiveness. To test the additional hypothesis that calorie restriction would reverse age-associated changes in leptin responsiveness, we compared food intake, body fat, and plasma hormone levels among young (Y), middle-aged (MA), and CR mice infused with leptin or vehicle.
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METHODS |
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Animals
The Institutional Animal Care and Use Committee of Albany Medical College approved all animal procedures for these experiments. All mice were purchased through the Office of Biological Resources and Resource Development of the National Institute on Aging. Mice were housed individually in an American Association for Accreditation of Laboratory Animal Care-approved, viral antibody-free facility and allowed to acclimatize forExperiment 1
Y (2 mo old) and MA (11-12 mo old) male C57BL/6 mice were implanted with subcutaneous osmotic minipumps (Durect, Cupertino, CA) under isoflurane anesthesia. Minipumps delivered either phosphate-buffered saline vehicle (PBS) or 0.15, 0.5, or 1.5 µg/h murine leptin (donated by Amgen, Thousand Oaks, CA). All mice were implanted with pumps on the same day. Body weight and ad libitum (ad lib) intake of standard NIH-31 diet were measured daily within 2 h of the same time each day throughout the experiment. Body weight was corrected for the weight of the minipump. Mice were killed without fasting in the morning after 3 or 10 days of infusion to assess early and sustained responses to leptin, respectively. Mice were bled for plasma hormones by retroorbital puncture and immediately killed by decapitation for tissue collection.Experiment 2
Experiment 2 included an additional group of MA mice from the National Institute on Aging colony that had been calorie restricted (CR) from 16 wk of age to 60% of normal intake. This restriction paradigm has been shown to increase longevity and is used as a standardized procedure at the National Institute on Aging Aged Rodent Colonies to provide calorie-restricted cohorts for aging research (27). Y, MA, and CR mice were infused subcutaneously with either PBS or 0.25 µg/h leptin for 4 days. This intermediate dose and time regimen was designed, on the basis of findings in experiment 1, to elevate plasma leptin levels but minimize catabolism in MA mice. Mice were bled for pretreatment plasma levels of leptin and insulin by retroorbital puncture (~100 µl) under anesthesia immediately after minipump implantation, between 3 and 4 h after lights-on. Mice were bled again on infusion day 2 within 1 h of lights-off to determine circadian peak corticosterone levels. Food intake and body weight were measured daily as described above. CR mice were limited to 3 g each per day of an equicaloric, vitamin-fortified formulation of NIH-31 diet (27). CR rations were provided once per day within 2 h of lights-out to avoid disruption of circadian hormone rhythms (8). Y and MA mice had free access to standard NIH-31 diet. After 4 days of infusion, mice were killed without prior fasting and within 2 h of lights-on for blood and tissue collection, as described in Experiment 1.Assays
Plasma glucose, corticosterone, leptin, and insulin were measured using commercial kits as previously described (26). Body composition was analyzed using previously described modifications of published methods (26).Statistics
ANOVA and post hoc analyses were performed using Statview version 5.0 (SAS Institute, Cary, NC). Significance was defined as P < 0.05. Three-way ANOVA for age, treatment, and time (experiment 1) or two-way ANOVA for age and treatment (experiment 2) revealed significant effects of age or interaction of age and treatment for all variables examined except postinfusion leptin levels in experiment 1. Post hoc comparisons between each of three leptin infusions and PBS to assess leptin responsiveness were performed in Y and MA mice in experiment 1 by Dunnett's test. Post hoc comparisons among Y, MA, and CR mice receiving either leptin or PBS in experiment 2 were performed by t-test with Bonferroni correction for three comparisons: 1) and 2) to the other age groups receiving the same treatment and 3) between leptin and PBS in the same age group. Data are presented throughout as means ± SE; the lack of error bars indicates that the scale of the symbol or graph exceeded that of the error. Regressions of plasma leptin against body fat levels (Fig. 7) for Y and MA mice were evaluated by analysis of covariance (Statview). ![]() |
RESULTS |
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Experiment 1
Basal food intake and body weight. Before the experimental treatments, MA and Y mice differed significantly in absolute levels of body weight and food intake. MA mice weighed 31.09 ± 0.35 g (n = 39), whereas Y mice weighed 22.81 ± 0.4 g (n = 42; P < 0.05). Food intake in MA mice in this experiment was slightly but significantly lower than that in Y mice (Y, 3.88 ± 0.1 g/mouse; MA, 3.60 ± 0.08 g/mouse; P < .05); this difference was accentuated when food intake was normalized to body weight (not shown).
Leptin effects on food intake and body weight in Y and MA mice.
After 3 or 10 days of subcutaneous infusion of 0.15, 0.5, or 1.5 µg/h
murine leptin, plasma leptin levels were similar both between Y and MA
mice and over time (Table 1). Y mice
exhibited leptin-dependent decreases in food intake within 1 day of
treatment (Fig. 1, A1).
However, MA mice showed a delayed response to leptin, with no change in
food intake at any leptin dose by day 1 and reduced food
intake evident only by day 3 at the two higher doses (Fig.
1, A2). As has been reported previously for prolonged leptin infusion (11, 12), daily food intake returned to initial
levels in leptin-treated Y and MA mice by 10 days.
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Leptin effects on body composition in Y and MA mice.
We analyzed carcass fat and protein content to determine whether
changes in total food intake and body weight affected body composition.
PBS-treated MA mice had almost twice the body fat of the Y PBS group
(Fig. 3, A and B).
However, only Y mice exhibited dose-related decreases in carcass fat
content by 3 days of leptin treatment; these decreases were significant
at the two higher leptin doses (Fig. 3A). Although carcass
fat content tended to be lower at 3 days in MA mice infused with 0.15 µg/h leptin vs. PBS, body fat did not decrease progressively
with increasing leptin levels, and there were no significant effects of
any leptin dose on body fat at this time. By 10 days of infusion, as
with food intake and weight loss, both MA and Y mice showed similar
significant, leptin-induced fat loss (Fig. 3B).
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Experiment 2
Basal food intake, body weight, and hormone levels in Y, MA, and CR
mice.
We next tested whether calorie restriction, which minimizes weight gain
and improves insulin sensitivity in aging (3), would
improve leptin responsiveness in middle-aged mice. As in experiment 1, middle-aged mice fed ad libitum (MA) were
heavier than Y mice (Table 2).
Middle-aged mice that had been calorie-restricted (CR) according to
National Institute on Aging protocols (27) weighed
significantly less than MA but significantly more than Y mice (Table
2). In this experiment, initial daily food intake was slightly but
significantly higher in MA mice than in either Y or CR mice (Table 2).
However, normalized food intake in MA mice was still significantly
lower than that in Y mice and did not differ from that in CR mice
(Table 2). Corroborating studies of age-related adiposity and
hyperinsulinemia in other species (4, 10, 19),
postprandial plasma leptin and insulin levels 3-4 h into the light
(inactive) period were significantly higher in MA mice but were
comparable between Y and CR mice (Table 2).
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Leptin effects on body protein and food intake in Y, MA, and CR
mice.
We infused leptin at an intermediate dose and time (0.25 µg/h)
in experiment 2 to increase plasma leptin measurably but
minimize protein loss in MA mice. Data in Table
3 confirm that leptin infusion caused
comparable, significant elevations in plasma leptin in all three age
groups without affecting body protein (Table 3). This dose of leptin
caused similar, significant decreases in 4-day food intake in MA as
well as Y mice (Fig. 4B). In
contrast, leptin treatment did not reduce food intake in CR mice (Fig.
4B). Y mice actually ate significantly less, and MA mice ate
marginally less, during leptin infusion (P = 0.095 after Bonferroni correction) than did leptin-treated CR mice (Fig.
4B). Leptin effects were similar whether absolute or
normalized food intake was analyzed (not shown).
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Experiment 2: leptin effects on body fat in Y, MA, and CR mice. Leptin infusion also had divergent effects on body fat. In the PBS control groups, carcass fat content was similar between Y and CR mice and significantly lower than that in MA mice (Fig. 4A). However, only Y mice exhibited significant fat loss after leptin treatment (Fig. 4). Carcass fat in Y mice was also significantly lower than that in MA and CR mice after leptin treatment (Fig. 4A).
Leptin effects on hormones and glucose in Y, MA, and CR mice.
Young mice were also the only group to show lower levels of glucose and
insulin in association with leptin-induced decreases in food intake and
body fat (Fig. 5). Morning plasma glucose
and insulin levels (Fig. 5) were marginally lower in leptin- vs.
PBS-treated Y mice (P = 0.0504 and 0.098, respectively,
after Bonferroni correction). Despite similar leptin-induced decreases
in food intake in MA mice, changes in plasma glucose and insulin did
not even approach significance in this group (P > 0.5 before correction). Morning plasma insulin in leptin-treated Y mice was
also marginally lower than that in leptin-treated CR and MA mice
(P = 0.07 after correction).
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Experiments 1 and 2: relationship of endogenous plasma leptin to body fat in Y and MA mice
To determine whether the endogenous leptin signal might also be altered in middle age, we performed linear regression of plasma leptin against carcass fat by using values from each PBS-treated Y and MA mouse in experiments 1 and 2. There was a significant linear correlation between plasma leptin and body fat levels in Y mice (Fig. 7). However, plasma leptin tended to be lower and was not significantly correlated with body fat in middle-aged mice (Fig. 7).
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DISCUSSION |
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In support of our first hypothesis and extending evidence for age-related leptin resistance (22, 25) to middle age, we have shown that leptin signaling is impaired during the period of midlife weight gain in male C57BL/6 mice (27). We found several deficits in MA mice, including slower and less selective metabolic responses to leptin, as well as a lack of correlation between body fat and endogenous plasma leptin levels. Contrary to our second hypothesis, we found that although calorie restriction reduces body fat, leptin, and insulin levels, it actually increases resistance to the appetite-suppressing effects of leptin and does not improve metabolic responses in middle-aged mice.
Slower Responses to Leptin in MA Mice
Results from experiment 1 indicated that, although leptin rapidly reduced food intake and body fat in Y mice, leptin had no consistent effect on these end points in MA mice for up to 3 days. Delayed leptin effects are unlikely to be due to slower equilibration of leptin levels in the larger MA mice, because we have found that these infusion rates significantly increase plasma leptin within 2 h in even bigger (>40 g) mice (not shown). Experiment 2 confirmed impaired fat mobilization in MA mice, even after comparable, leptin-induced decreases in food intake (Fig. 4). A variety of hypothalamic and adipocyte responses to leptin have been shown to be impaired in 24-mo-old rats (20, 22, 23, 25). However, because body weight declines after this age in rats (22), it was unclear whether leptin resistance was relevant to the midlife weight gain that decreases longevity (4, 7, 14, 24). The delayed and reduced leptin responsiveness we have found at an earlier life stage could account for weight gain at middle age by allowing calorie intake and storage to outpace compensatory responses to increasing fat mass.Less Selective Metabolic Responses to Leptin in MA Mice
Although leptin resistance of MA mice appeared to be overcome by longer treatment times, leptin-induced decreases in food intake, body weight, and body fat occurred by 10 days in MA mice at the price of greater protein catabolism. Although Y and MA mice lost body protein at the highest leptin dose, this infusion increased plasma leptin almost 10-fold and was probably supraphysiological. However, MA mice also lost significant amounts of body protein after 10 days of infusion with 0.5 µg/h leptin, a dose that, in agreement with previous literature (12), did not affect body protein in Y mice. Thus MA mice not only respond to leptin more slowly but are also less able to mobilize fat preferentially after sustained leptin exposure. The etiology of this leptin resistance remains to be elucidated but could include loss (6) or impaired intracellular signaling (23) of leptin-responsive hypothalamic neurons, as well as adipocyte resistance to leptin-induced increases in sympathetic stimulation (20).No Correlation Between Body Fat and Endogenous Plasma Leptin in MA Mice
The correlation between plasma leptin and body fat is lost in older humans, suggesting that increasing age-related adiposity represents a compensatory response to disproportionately low leptin levels (16). In agreement with the human data (16), we found that body fat was not a significant predictor of plasma leptin levels in MA mice. Deficits in maintaining plasma leptin levels are clearly not the only cause of altered weight regulation in MA mice, since MA responses were impaired even after plasma leptin was increased by infusion. The finding that transgenic mice overproducing leptin still get fat with age (18) also suggests that leptin resistance is a key factor in midlife weight gain.CR Mice Do Not Respond to Leptin Infusions Inhibiting Food Intake in MA Mice
We have further shown that calorie restriction, which minimizes weight gain and improves insulin sensitivity in aging (4), actually increases resistance to the appetite-suppressing effects of leptin. Infusion of a moderate leptin dose (0.25 µg/h) that reduced 4-day food intake in MA mice had no effect in CR mice. Suppression of MA food intake was not inconsistent with experiment 1, since 4 days of treatment probably augmented leptin-related differences emerging by 3 days; calculation of 4-day cumulative food intake for experiment 1 did show significant decreases in the 0.5 and 1.5 µg/h MA groups (not shown). The failure of leptin to suppress food intake in CR mice was not due to restricted food availability during the infusion, since leptin-treated Y mice ate significantly less than did leptin-treated CR mice (Fig. 4). Initial levels of food intake in CR mice were also similar to those in MA mice when expressed relative to body weight (Table 2) or lean body mass (calculated as carcass weight minus fat weight; not shown). Therefore, CR food rations during the experiment were neither so absolutely nor disproportionately low that further decreases were impossible.Metabolic Responses to Leptin Are Not Improved in CR Mice
Like their MA counterparts, CR mice did not decrease body fat or plasma insulin as efficiently as Y mice after leptin treatment. CR mice were not so lean that further reductions were impossible, since Y mice were as lean and yet underwent further, leptin-induced loss of body fat. MA and CR mice not only failed to exhibit leptin-dependent decreases but also had significantly higher levels of body fat and marginally higher levels of plasma insulin than Y mice after leptin treatment. Significant leptin-induced loss of body fat might have been expected not only in MA mice, which decreased food intake, but also in CR mice, because leptin can stimulate fat mobilization independently of food intake (11). These results suggest that leptin-induced fat mobilization in MA mice is not improved by calorie restriction. Leptin effects on glucose homeostasis in Y mice, which were probably underrepresented because mice were not fasted, could have been due to greater loss of body fat as well as leptin inhibition of food intake. However, because leptin can enhance insulin action independently of changes in food intake and fat deposition (5, 9), the lack of leptin effects on insulin in CR as well as MA mice also suggests that calorie restriction may not fully reverse age-related insulin resistance.Incomplete Leptin Suppression of CR Plasma Glucocorticoid Levels
As expected from previous literature (2, 13, 17), CR mice had markedly higher circadian peak corticosterone levels that tended to be reduced by leptin infusion. However, even after leptin treatment, plasma corticosterone was still significantly higher in CR vs. MA mice. Because we have previously shown that corticosterone levels in this range can block leptin effects on food intake and body fat (26), higher corticosterone levels in CR mice may contribute to their relative refractoriness to leptin. Notably, leptin treatment did not reduce trough or peak corticosterone levels in Y or MA mice. We suspect that the reported inhibition of circadian glucocorticoid secretion by leptin infusion (1) was confounded by stress effects on adrenocortical activity.We have shown that MA mice exhibit impaired responses to leptin that are not improved or, in the case of food intake, are further disrupted by calorie restriction. The failure of leptin action in MA mice is consistent with the likelihood that excess body weight reflects leptin resistance rather than leptin deficiency (11) and affirms the anticipated difficulty of achieving significant public adherence to lifelong calorie restriction. Identifying the factors causing leptin resistance during aging and calorie restriction will be necessary to make dietary restraint a more tolerable, if not avoidable, strategy for healthy aging.
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ACKNOWLEDGEMENTS |
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I am grateful to Brian D. Mooney and Rebecca Rokow-Kittell for technical assistance. This study was supported in part by National Institute on Aging Grant AG-18241 to the author.
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FOOTNOTES |
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Address for reprint requests and other correspondence: Lauren Jacobson, Center for Neuropharmacology and Neuroscience, Albany Medical College, MC-136, Albany, NY 12208 (E-mail: JACOBSL{at}mail.amc.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. Section 1734 solely to indicate this fact.
10.1152/ajpendo.00495.2001
Received 6 November 2001; accepted in final form 20 December 2001.
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