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Disruption of the Relationship between Fat Content and Leptin Levels with Aging in Humans¹

Division of Endocrinology (N.M., K.S.N.) and Section of Biostatistics (P.O.), Mayo Clinic, Rochester, Minnesota 55905

Address all correspondence and requests for reprints to: K. Sreekumaran Nair, M.D., Ph.D., Mayo Clinic, Endocrine Research Unit, 5–164 West Joseph, Rochester, Minnesota 55905. E-mail: nair.sree{at}mayo.edu

	Abstract

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Leptin released from adipose tissue is believed to participate in a negative feedback loop regulating appetite, and malfunction of this mechanism could lead to obesity. We measured plasma leptin and body composition (dual energy x-ray absorptiometry) in 70 healthy subjects, divided into 3 age groups (young, 25 ± 1 yr; middle-aged, 53 ± 1 yr; old, 70 ± 1 yr), while on a 5-day weight-maintaining diet. Pairwise correlations were assessed using product-moment correlation, and regression analysis was used to evaluate relationships between leptin and other variables.

Leptin concentrations and relative body fat content were correlated in young females (r = 0.71; P = 0.009) and in young males (r = 0.76; P = 0.007), but not in the combined middle-aged and elderly groups (r = 0.19; P = 0.36 and r = 0.19; P = 0.38 in females and males, respectively). Regression analysis showed a clear correlation between circulating leptin and relative fat mass in the young subjects (P = 0.0001), but not in the older subjects (P = 0.199).

We conclude that body fat content in young subjects correlates with plasma leptin in both genders, whereas this relationship is disrupted in elderly subjects, thus possibly contributing to the obesity occurring with age.

	Introduction

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LEPTIN released from adipocytes (1) is thought to play a key role in the regulation of energy intake, thermogenesis, and body weight (2). Experiments in rodents have shown that administration of leptin induces weight loss due to a combination of decreased food intake and increased energy expenditure (3, 4). Studies in humans have found a strong positive correlation between serum leptin concentrations and body fat (5, 6) together with overexpression of leptin genes (messenger ribonucleic acid) in adipose tissue from obese subjects (7, 8). In addition, a several-fold increase in leptin levels has been reported in women compared to men regardless of reproductive hormone status and total body fat content (5, 6, 9). It has been proposed that these gender differences could relate to dissimilarity of regional body fat composition (9). Resistance to the metabolic actions of leptin has been reported in certain obese mouse strains (10), but it is also possible that failure of adipocytes to secrete sufficient amounts of leptin could lead to relative hypoleptinemia and obesity.

In humans the aging process is associated with a progressive gain of fat mass and loss of muscle mass and performance (11, 12). It has been found that, in general, elderly people have lower circulating levels of leptin than the young (13), and it is thus possible that aging may distort the direct association between leptin and fat mass.

We, therefore, proposed to test the hypothesis that the association between body fat and leptin is disturbed with advancing age. We also investigated whether the variation in leptin concentrations between genders may be attributed to differences in regional body fat distribution.

	Experimental Subjects

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Seventy healthy volunteers (20–79 yr old) were studied. They were participants in a larger study protocol, assessing the mechanism of muscle wasting in aging; results from this study have been reported in part previously (14). Only healthy subjects with normal physical examination and normal biochemical screening (including kidney and liver function) were recruited. All subjects had a normal fasting glucose concentrations on two separate occasions.

Postmenopausal women receiving estrogen replacement therapy, subjects taking ß-adrenergic blockers, and subjects exercising more than twice a week for 20 min each time were excluded. Detailed subject data are given in Table 1. The subjects were divided into 3 age groups: young (25 ± 1 yr), middle-aged (53 ± 1 yr), and old (70 ± 1 yr); each age group consisted of 12 women and 11 or 12 men.

	Materials and Methods

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Body composition

On day 2, whole body and regional body composition were determined in all subjects in the basal postabsorptive state employing dual energy x-ray absorptiometry (model DPX-L, Lunar Corp., Madison, WI) (15), and fat and lean tissue contents were calculated. Both whole body and regional measurements were made, and separation of truncal and peripheral tissues was made on the basis of arbitrary antero-posterior division lines through the shoulder joints and through the femoral necks.

Circulating hormones and metabolites

Plasma concentrations of leptin were measured using a commercial RIA for human leptin (Linco Research, St. Charles, MO). Plasma samples (100 µL) were incubated overnight with [¹²⁵I]leptin and leptin antibody overnight. The next day, the bound fraction was counted after precipitation (within-assay coefficient of variation, <5%). Serum concentrations of insulin were determined in a chemiluminescent sandwich assay (Sanofi Diagnostics, Chaska, MN), and serum levels of total testosterone and free testosterone were both assayed using commercial RIAs (Incstar Corp., Stillwater, MN; Diagnostic Systems Laboratories, Webster, TX). Circulating plasma levels of free fatty acids were measured by an enzymatic calorimetric method (Wako Chemicals, Richmond, VA).

Statistical analysis

Pairwise correlations between quantitative characteristics were evaluated using product-moment correlation. Regression analysis was used to assess the relationship between leptin and multiple explanatory variables. A log transform was used for leptin values because the data were highly skewed. Data from old and middle-aged subjects were combined because they were quite similar and to increase the sample size. Data from the resulting "older" group were analyzed separately from those from the young because variability was considerably larger in the older group. The associations with leptin were also found to be qualitatively different between age groups.

A stepwise regression analysis (stepping up, using 0.05 as the criteria for inclusion in the model) was used to identify associations between the larger group of explanatory variables and leptin. Log transforms were used for insulin, free fatty acids, total testosterone, and free testosterone. Testosterone was retained in the stepwise selection only if it achieved statistical significance with gender also in the model.

Unless specified otherwise data given below are the mean ± SEM.

	Results

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The clinical characteristics, anthropometric measures, and hormone levels of the participants are given in Tables 1 and 2. Their body weights ranged from 47.3–107.5 kg, and body mass indexes ranged from 19.0–35.0 kg/m². Total body fat content determined by dual energy x-ray absortiometry scans ranged from 7.3–40.4 kg, and truncal fat content ranged from 3.3–21.0 kg. Circulating leptin concentrations were 16.2 ± 1.5 (range, 3.2–35.3) ng/mL in women and 4.4 ± 0.5 (range, 2.0–13.8) ng/mL in men, and serum insulin concentrations were comparable among groups. Plasma concentrations of free fatty acids were 0.590 ± 0.03 mmol/L in women and 0.470 ± 0.03 mmol/L in men.

Regression analysis relating leptin to total body fat and gender indicated that, as expected, leptin levels were higher in females (Table 3). However, although an association with total fat was found among young subjects, no association was observed in the older cohort (Table 3 and Figs. 1 and 2).

View larger version (9K): [in this window] [in a new window]   Figure 1. Relationship between plasma leptin concentrations and relative body fat content in the middle-aged and the old subjects (r = 0.19; P = 0.36 in females and r = 0.19; P = 0.38 in males). • and ——, Males; and - - -, females.

View larger version (8K): [in this window] [in a new window]   Figure 2. Relation between plasma leptin concentrations and relative body fat content in young subjects (r = 0.71; P = 0.009 in females and r = 0.76; P = 0.007 in males). • and —-, Males; and - - -, females.

When testosterone was included in the analysis (and gender excluded), there was a significant negative association between total testosterone and leptin in the older cohort (r = 0.4257; SE = 0.0578; P < 0.001).

	Discussion

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The present data, while confirming previous reports of a strong positive correlation between plasma leptin concentrations and body fat content in younger subjects, provide novel evidence that this intimate association is disrupted in older subjects. Compared to those in men, leptin levels are elevated in women; however, the lack of relationship between circulating leptin and body fat with advancing age applies equally to both genders. Plasma leptin concentrations were not additionally associated with regional body fat distribution.

Our results were obtained from a sample size of 70 subjects. It is, however, unlikely that the lack of any significant association between circulating leptin and body fat in the older subjects relates to the relatively small sample size, as the 95% confidence interval for the coefficient in the model is -0.945 to 4.399 x 10^-3.

Based on the observation that expression of the ob gene varies between fat depots (16), it has been proposed that gender differences in plasma leptin could be caused by differential regional patterns of fat deposition (9). This hypothesis is not supported by our observation that circulating leptin is determined by total, rather than regional, fat mass. Two recent studies have also reported lack of any intrinsic association between plasma leptin and the size of regional fat depots in either African-American women (17) or Japanese-American women and men (18).

Most subjects included in previous studies have been relatively young. Two studies have shown that serum leptin concentrations, when corrected for percent body fat, are inversely correlated to age (13, 19), but the question of whether the correlation between leptin and body fat is preserved in the elderly was not addressed. By showing that by middle age and beyond an increased fat mass is not paralleled by high levels of circulating leptin, our results suggest that impaired secretion or, less likely, increased clearance could be of pathophysiological importance for obesity. The very recent observation that low plasma leptin concentrations precede weight gain in Pima Indians also supports this idea (20).

It has been proposed that obesity in humans may be caused by resistance to leptin (2). Leptin resistance has been demonstrated in the diabetic (db/db) mouse due to mutations in the leptin receptor gene (2, 10), and the finding of relatively decreased levels of leptin in the cerebrospinal fluid compared to the circulation in obese subjects suggests that defective blood-brain transportation may be involved in humans (21).

The present results are still compatible with the suggestion that leptin resistance may be important in the pathogenesis of obesity, in particular in the young, but additionally suggest that relative leptin deficiency may gain increasing significance with advancing age. By implicating a pathophysiological role of leptin deficiency, our data provide a rationale for future therapeutic assessment of leptin, leptin analogs, or perhaps leptin secretogogues in the aging population prone to obesity.

The possibility that the observed age-related weakening of the relationship between leptin in the circulation and the size of body fat stores could be caused by an increased fat mass per se rather than age seems unlikely. Although the elderly subjects in our study also tended to be more obese, there was a considerable overlap in relative fat mass between young and older subjects. Furthermore, many previous studies have shown that the association between body fat and leptin gene expression and leptin concentrations persists in varying degrees of obesity (5, 6, 7, 8, 13, 17, 18).

It is unclear which mechanisms cause the age-dependent alterations in the regulation of circulating leptin concentrations. In the obese (ob/ob) mouse, leptin secretion is absent due to a mutation (1, 2), but as the lack of association between leptin and body fat occurring with age in humans appears to be a general phenomenon, there is little reason to suspect any specific genetic cause. More studies, including stimulation of leptin secretion, are needed to determine the nature of the low leptin levels in terms of capacity of the adipocytes to secrete leptin, protein binding and composition in the circulation, and leptin clearance.

Another interesting observation in our study is that there is a negative correlation between circulating levels of leptin and testosterone among the older subjects. It has been shown that leptin may be a key link between energy stores (or nutritional status) and the reproductive axis (22), and it is intriguing that our observation of a loss of correlation between leptin in the circulation and body fat stores in both men and women after middle age coincides with menopause in women and the age-related decline in testosterone in men.

Based on our data, we conclude that, unlike in the young subjects (20–40 yr), in older subjects (>45 yr) there is a lack of correlation between circulating leptin levels and body fat content. This suggests an impairment of the feedback between peripheral fat stores and appetite regulation in the central nervous system in elderly subjects, an impairment that may contribute to the increased prevalence of obesity with aging and interact with age-related changes in sex hormone levels.

	Acknowledgments

 
We gratefully acknowledge support from Jody Utton and Maureen Bigelow in performing the study and collecting the data, Dr. George Klee (Immunochemistry Core Laboratory) for performing the RIAs, and the helpful suggestions from Drs. Robert Rizza and Michael Jensen.

	Footnotes

 
¹ This work was supported by USPHS NIH Grants RO1-AG-0973, RO1-DK-94020–1, DK-50456, RR-00585, and RR-109.

Received July 9, 1997.

Revised September 3, 1997.

Accepted November 18, 1997.

	References

Top Abstract Introduction Experimental Subjects Materials and Methods Results Discussion References

 

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