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Effect of Short-Term Fasting on Free and Bound Leptin Concentrations in Lean and Obese Women

Departments of Pediatrics (M.L.) and Internal Medicine (J.F.H., S.K.), Washington University School of Medicine, St. Louis, Missouri 63110; and St. Bartholomew’s and The Royal London School of Medicine (S.W.C.), London, United Kingdom

Address all correspondence and requests for reprints to: Michael Landt, Ph.D., Department of Pediatrics, Washington University School of Medicine at St. Louis Children’s Hospital, One Children’s Place, St. Louis, Missouri 63110. E-mail: landt{at}kids.wustl.edu

Abstract

Plasma leptin exists in protein-bound and free forms, which may affect its hormonal bioactivity. Therefore, the relationship between bound and free leptin may be particularly important during physiological conditions that cause rapid alterations in total plasma leptin concentration, such as fasting. The purpose of this study was to evaluate the effect of short-term fasting on bound and free plasma leptin concentrations and leptin binding capacity (a measure of plasma leptin-binding protein content) in lean and obese women. Six lean (body mass index, 21 ± 1 kg/m²) and 6 abdominally obese (BMI, 36 ± 1 kg/m²) women were studied after 14 h and 22 h of fasting. Although total plasma leptin concentration was more than 6-fold greater in obese (45.4 ± 7.6 µg/liter) compared with lean (7.4 ± 1.0 µg/liter) women at 14 h of fasting (P < 0.05), the percentage of leptin in the bound form was greater in lean than obese subjects (29 ± 2% vs. 12 ± 3%; P < 0.05). Arterial total, free, and bound plasma leptin concentrations all declined between 14 h and 22 h of fasting in both lean and obese groups, but the relative decline of these fractions was greater in lean (36 ± 4%, 60 ± 9%, and 51 ± 13%, respectively) than in obese (19 ± 5%, 21 ± 8%, and 12 ± 7%, respectively) subjects (all P < 0.05). In contrast, leptin binding capacity was unchanged. The percentage of total plasma leptin present in bound form was constant between 14 h and 22 h of fasting in lean subjects and increased slightly but significantly in obese subjects. These data demonstrate that both free and bound fractions of leptin in plasma decrease quickly in response to energy restriction, but the decline is blunted in abdominally obese compared with lean women. In addition, the equilibrium between bound and free leptin fractions is maintained during brief fasting and is not regulated by leptin binding capacity.

LEPTIN, THE PROTEIN product of the ob gene, is produced primarily by adipose tissue and secreted into the circulation (1, 2). Many of leptin’s functions, such as regulating food intake (3, 4), energy expenditure, and reproductive function (5), are believed to require leptin transport across the blood-brain barrier and binding to specific receptors in the hypothalamus. A considerable portion of circulating leptin is bound to protein (6, 7); the amount bound to protein is affected by the degree of adiposity and nutritional state (6, 8). Although the physiological functions of bound and free leptin are not well understood, it has been hypothesized that leptin is more active in its free form because this form is present in cerebrospinal fluid (CSF) (9).

There is increasing evidence that leptin’s most important function may be in regulating the neuroendocrine response to fasting (5). In lean persons, plasma leptin concentrations decline markedly within the first 24 h of fasting (10), which may be important in regulating substrate metabolism and energy expenditure during early starvation (5). We have recently found that the normal reduction in adipose tissue leptin production and plasma concentration that occurs during early fasting is blunted in persons with abdominal obesity (11). This attenuated decline in plasma leptin levels may contribute to the alterations in the metabolic responses to fasting, particularly the blunted decline in sympathetic nervous system activity, observed in persons with abdominal obesity (12). However, the effect of brief fasting on bound and free fractions of leptin in persons with abdominal obesity is unclear.

The purpose of the present study was to evaluate the effect of brief fasting on free and bound arterial plasma leptin concentrations and leptin binding capacity in lean and abdominally obese women. In addition, samples were obtained from an abdominal vein that drains sc adipose tissue, which allowed characterization of the form (i.e. bound or free) of newly secreted leptin and to determine whether adipose tissue alters leptin binding capacity.

Materials and Methods

Subjects

Six lean women and six women with abdominal obesity (waist circumference, 115 ± 4 cm) participated in this study (Table 1). These subjects were composed of a subset of subjects who participated in a study investigating changes in total leptin production during fasting (11). The lean and obese groups were matched for fat-free mass, which was determined by dual-energy x-ray absorptiometry (Hologic QDR 1000/W; Hologic, Inc., Waltham, MA). Although the obese women were older than the lean women (38 ± 3 and 28 ± 3 yr, respectively; P < 0.05), all women were premenopausal. No subjects were taking any medications, and all were considered healthy, except for the presence of obesity. All subjects had normal fasting blood glucose concentrations and plasma lipid profiles. All subjects were weight-stable for at least 2 months before the study, which was performed within the first 2 wk of the follicular phase of their menstrual cycle. This study was approved by the Human Studies Committee and the General Clinical Research Center Scientific Advisory Board of Washington University School of Medicine. Informed consent was obtained from all subjects before their participation.

Subjects were admitted to the Clinical Research Center of Washington University School of Medicine in the evening before the study. At 1800 h, subjects ate a standardized meal containing 12 kcal/kg body weight for lean subjects and 12 kcal/kg adjusted body weight for obese subjects (adjusted body weight = ideal body weight + [(actual body weight - ideal body weight) x (0.25)]). Ideal body weight was defined as the midpoint of the medium frame of the 1983 Metropolitan Life Insurance Table. An adjusted body weight was used to help normalize energy intake with energy requirements between lean and obese subjects by assuming that approximately 25% of excess body weight in our obese subjects was composed of fat-free mass. At 2000 h, all subjects ingested a defined liquid formula snack containing 240 kcal (Ensure; Ross Laboratories, Columbus, OH). After this snack, all subjects fasted until completion of the study the following day.

The next morning a radial artery was catheterized for arterial blood sampling, and a 22-gauge catheter was inserted into an abdominal vein to sample blood draining sc adipose tissue (13). The abdominal catheter placement was successful in only four lean and four obese women, therefore, all regional adipose tissue data were limited to these subjects. Artery and abdominal vein samples were obtained simultaneously every 5 min (four samples) between 0945 and 1000 h (13 h, 45 min and 14 h of fasting) and again at 1745–1800 h (21 h, 45 min and 22 h of fasting) to determine total, free, and bound leptin concentrations. The average leptin concentrations in the four blood samples taken between 0945 and 1000 h and again between 1745 and 1800 h represented 14 h and 22 h of fasting values, respectively.

Analytical procedures

Total plasma leptin concentration was measured by RIA, as described previously (14). Plasma free and bound leptin concentration and leptin binding capacity were measured by a recently developed procedure (15), which uses high-performance liquid chromatography separation of free and bound forms, and quantification of leptin content in chromatography fractions with a high-sensitivity leptin RIA. The chromatography method recovers about 65% of the total leptin in the free and bound fractions. Therefore, the sum of the free and bound leptin fractions does not equal total plasma leptin concentration measured by RIA. Leptin binding capacity, which is an indirect measure of the overall concentration of leptin-binding proteins in plasma, was measured as described previously (15).

Calculation

Percentage of leptin in the bound-state (% bound-leptin) was calculated as the bound plasma leptin concentration divided by the sum of free and bound plasma leptin concentrations, multiplied by 100.

Statistical analysis

Student’s t test for normally distributed data and the Mann-Whitney rank sum test for nonnormally distributed data were used to evaluate differences between groups, and a paired Student’s t test for paired samples was used to evaluate differences between 14 and 22 h of fasting within groups. A P value of 0.05 or less was considered to be statistically significant. All results are presented as means ± SE.

Results

Arterial plasma leptin concentrations

Total, free, and bound leptin concentrations were greater in obese than in lean subjects at 14 h and 22 h of fasting (P < 0.05) (Table 2). All leptin fractions decreased between 14 h and 22 h of fasting in both groups (Table 2). However, the reduction in bound-leptin concentration in obese subjects did not reach statistical significance (P = 0.10), which may reflect a Type II statistical error. The relative reductions in total, free, and bound leptin concentration were significantly greater (P < 0.05) in lean than obese subjects (Table 2), and the relative decline in free leptin was similar to the fall in bound leptin concentration (Table 2). In contrast to the significant decline in total, free, and bound leptin concentrations with fasting, arterial leptin binding capacity was not significantly different between 14 and 22 h of fasting in either the lean (1.8 ± 0.1 µg/liter vs. 1.7 ± 0.2 µg/liter, P > 0.05) or obese (3.5 ± 0.4 µg/liter vs. 3.6 ± 0.6 µg/liter, P > 0.05) groups, respectively.

At 14 h of fasting, the percentage of arterial plasma leptin in the bound form was more than 2-fold greater (P < 0.05) in lean than in obese subjects (28.9 ± 2.1% vs.12.0 ± 2.5%, respectively). Continued fasting to 22 h did not significantly alter the relative contribution (percentage) of bound leptin in lean subjects (33.8 ± 4.2%), but there was a small but significant increase in obese subjects (to 13.5 ± 2.9%, P < 0.05). Thus, despite relatively large magnitude decreases in absolute concentrations of bound leptin in both lean and obese subjects (Table 2), there was little change in the percent bound leptin in either lean or obese subjects.

Abdominal vein leptin concentrations and binding capacity

Total, free, and bound plasma leptin concentrations were greater in abdominal vein than in radial artery samples in both lean and obese subjects (Table 3). However, the abdominal vein values were not statistically significantly different than the arterial values in the lean group. We combined data from lean and obese subjects because of the similar trends and the small number of subjects in each group (Table 3). The combined data demonstrated that total, free, and bound leptin concentrations were significantly greater in abdominal vein than in radial artery samples (29 ± 8%, 51 ± 20%, and 30 ± 12% for total, free, and bound leptin concentrations, respectively; all P < 0.05). In contrast, leptin binding capacity was similar in abdominal vein and artery samples in lean and obese subjects, as well as the combined data (Table 3).

When results for both lean and obese subjects at 14 h and 22 h were combined, there were strong correlations of bound leptin concentrations with paired concentrations of both total leptin (r² = 0.69, P < 0.05) and free leptin (r² = 0.79, P < 0.05). These correlations were improved by log transforming the total leptin concentrations (r² = 0.77; P < 0.05; Fig. 1A) and the free leptin concentrations (r² = 0.89, P < 0.05; Fig. 1B).

View larger version (14K): [in this window] [in a new window]   Figure 1. Relationships between log10-transformed total and bound plasma leptin concentrations (A) and between log10-transformed free and bound plasma leptin concentrations (B), in lean and obese subjects. Data are from both 14 h and 22 h of fasting, and arterial and abdominal vein blood samples.

The reduction in plasma leptin concentration during early starvation is an essential component of the neuroendocrine response to fasting (5). In persons with abdominal obesity, a blunted decline in plasma leptin concentration (11) may contribute to some of the alterations in the metabolic response to fasting (12). Moreover, variations in the proportion of leptin associated with plasma-binding proteins may also regulate leptin bioavailability and bioactivity (6, 7). The results of the present study demonstrated that the decline in both bound- and free-leptin concentrations were blunted in persons with abdominal obesity during short-term fasting. However, leptin binding capacity was not affected by fasting in lean or obese women. Because leptin binding capacity reflects the concentration of plasma proteins that associate with leptin (15), these data suggest that brief fasting does not alter leptin-binding protein concentration in lean or abdominally obese women. The proportion of leptin bound to plasma proteins remained constant (or nearly so) between 14 and 22 h of fasting, despite comparatively large decreases in absolute concentrations of bound leptin in both lean and obese subjects. The maintenance of a constant proportion of bound leptin suggests that leptin binds to plasma proteins in dynamic equilibrium, so that decreased leptin levels during fasting results in dissociation of leptin from binding proteins.

It has been hypothesized that the free form of leptin is more physiologically active than the bound form (9), which is true for many other peptide hormones (16). Binding proteins can function as a reservoir for an inactive hormone, so an acute decrease in hormone production may be countered by dissociation from binding proteins to attenuate the decline in active (i.e. free) hormone concentration. The results of the present study support the notion that leptin binding acts as an additional regulator of leptin action. We found that the amount of leptin circulating in the bound form correlated with total plasma leptin concentration and changed in concert with alterations in plasma free-leptin concentration. Bound-leptin concentration decreased when total leptin concentration declined during fasting, making more free leptin available in the circulation. In addition, bound-leptin concentration was greater in abdominal venous blood than in the arterial circulation, indicating that leptin binding attenuates the increase in abdominal vein free leptin caused by leptin secretion from adipose tissue. In contrast, Sinha et al. (6) reported that the changes in free-leptin concentration during fasting and refeeding was not associated with a significant change in bound-leptin concentration (6). However, the relative decline in bound leptin concentration during fasting reported by Sinha et al. (6) (37% and 17% in lean and obese subjects, respectively) was similar to the decline we observed in our subjects (45% and 12% in lean and obese subjects, respectively). The small number of subjects studied by Sinha et al. (6) (three lean and three obese subjects) made it difficult to detect a statistically significant reduction in bound-leptin concentrations in their study.

The site and regulation of leptin-binding protein production are unknown. The results of the present study suggest that adipose tissue does not produce leptin-binding proteins, although these experiments were based on abdominal vein sampling and, therefore, only sc fat was studied. If adipose tissue produced leptin-binding proteins, leptin binding capacity, which reflects the concentration of leptin-binding proteins in plasma, should have been greater in abdominal vein than arterial samples. However, leptin binding capacity was identical in abdominal vein and radial artery blood. In contrast, total, free, and bound leptin concentrations were greater in abdominal vein than in radial artery blood samples because of leptin production by adipose tissue. The increase in bound leptin at static levels of binding capacity adds further evidence that bound leptin concentrations are determined by the dynamic equilibrium between the free and bound forms, so that adipose tissue secretion of free leptin into the bloodstream will increase the association between secreted leptin and binding proteins. Therefore, binding proteins are probably only passive regulators of the amount of bound leptin in plasma.

In this study, we evaluated the effect of brief fasting on plasma leptin metabolism. Therefore, the observations made during this short interval of fasting (14–22 h) may not necessarily apply to more prolonged fasting. Extending the duration of fasting would have caused greater decreases in total, free, and bound plasma leptin concentrations and might have generated changes in leptin binding capacity. Longer-term fasting studies are needed to evaluate these possibilities.

The higher initial plasma leptin concentrations in obese compared with lean subjects was due to a disproportionate increase in free over bound leptin. Our findings are consistent with data reported by Sinha et al. (6), indicating that increased adiposity is associated with an increased concentration of free leptin. These findings support the notion that obese persons are resistant to the effects of leptin in regulating energy balance and that differences in leptin binding are not responsible for differences in leptin action. There are several potential causes for leptin resistance in obesity. Reduced transport of leptin across the blood-brain barrier would decrease leptin concentration at its primary site of action in the hypothalamus. This leptin transport system has been found to be saturable in humans (17) and the efficiency of leptin transport from blood to the CSF is reduced in obese persons (18). In addition, variations in hypothalamic leptin-receptor density and affinity for leptin, or postreceptor effects may also alter the metabolic responses to leptin (19). It is likely that variation in leptin sensitivity among humans depends on the combined regulation of leptin transport into CSF, as well as leptin receptor and postreceptor function.

Acknowledgments

We thank the nursing staff of the General Clinical Research Center for help in performing the experimental protocols.

Footnotes

This study was supported by NIH Grants DK-37948, RR-00036 (General Clinical Research Center), AG-13629 (Claude Pepper Older American Independence Center), AG-00078 (Institutional National Research Service Award), and DK-56341 (Clinical Nutrition Research Unit).

Abbreviations: CSF, Cerebrospinal fluid.

Received December 6, 2000.

Accepted April 20, 2001.

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