X/97/$03.00/0 Vol. 82, No. 4 Journal of Clinical Endocrinology and Metabolism Copyright 1997 by The Endocrine Society

0021-972X/97/$03.00/0 Vol. 82, No. 4 Journal of Clinical Endocrinology and Metabolism Printed in U.S.A. Copyright 1997 by The Endocrine Society COMMENTS The Metabolic Significance of Leptin in Humans: Gender-Based Differences in Relationship to Adiposity, Insulin Sensitivity, and Energy Expenditure* ADELE KENNEDY, THOMAS W. GETTYS, PATRICIA WATSON, PENNY WALLACE, ELIZABETH GANAWAY, QIN PAN, AND W. TIMOTHY GARVEY Departments of Medicine (A.K., T.W.G., Pa.W., Pe.W., E.G., W.T.G.), Biochemistry and Molecular Biology (T.W.G.), and Biometry and Epidemiology (Q.P.), Medical University of South Carolina, and the Charleston Veterans Affairs Medical Center, Charleston, South Carolina 29425 LEPTIN IS a 16-kDa plasma protein synthesized in adipose tissue and was identified as the mutant gene product in ob/ob mice (1, 2). Mice inheriting a copy of this gene from both parents exhibit marked obesity, hyperphagia, glucose intolerance, insulin resistance, low energy expenditure, and sterility (2a); exogenous recombinant leptin injected into these mice reverses these metabolic parameters (2 5). A further monogenic cause of obesity has been identified in the db/db mouse (5). When present in the same genetic background, ob and db genes produce similar metabolic and behavioral phenotypes. db/db mice, however, fail to respond to Received August 15, 1996. Revision received October 14, 1996. Rerevision received November 26, 1996. Accepted December 5, 1996. Address all correspondence and requests for reprints to: W. Timothy Garvey, M.D., Division of Endocrinology, Diabetes, and Medical Genetics, Medical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina 29425. E-mail: garveywt@musc.edu. * This work was supported by NIH Grants DK-38765, DK-47461, and DK-42486; a Merit Review grant from the Department of Veteran Affairs; and the General Clinical Research Center at the Medical University of South Carolina (Grant M01-RR001070). ABSTRACT Leptin is an adipocyte-derived hormone that interacts with a putative receptor(s) in the hypothalamus to regulate body weight. The relationship of leptin to metabolic abnormalities associated with obesity together with hormonal and substrate regulation of leptin have not been extensively studied. Therefore, 116 subjects (62 men and 54 women) with a wide range of body weight [body mass index (BMI), 17 54 kg/m 2 ] were characterized on a metabolic ward with regard to body composition, glucose intolerance, insulin sensitivity, energy expenditure, substrate utilization, and blood pressure. Eighty-five of the subjects had normal glucose tolerance (50 men and 35 women), and 31 had noninsulin-dependent diabetes mellitus (12 men and 19 women). In both men and women, fasting leptin levels were highly correlated with BMI (r 0.87 and r 0.88, respectively) and percent body fat (r 0.82 and r 0.88, respectively; all P 0.0001). However, men exhibited lower leptin levels at any given measure of obesity. Compared with those in men, leptin levels rose 3.4-fold more rapidly as a function of BMI in women [leptin 1.815 (BMI) 31.103 in women; leptin 0.534 (BMI) 8.437 in men] and 3.2 times more rapidly as a function of body fat [leptin 1.293 (% body fat) 24.817 in women; leptin 0.402 (% body fat) 3.087 in men]. Hyperleptinemia was associated with insulin resistance (r 0.57; P 0.0001) and high waist to hip ratio (r 0.75; P 0.0001) only in men. On the other hand, during the hyperinsulinemic euglycemic clamp studies, hyperinsulinemia acutely increased leptin concentrations (20%) only in women. There was no correlation noted between fasting leptin levels and either resting energy expenditure or insulin-induced thermogenesis in men or women (P NS). In stepwise and multiple regression models with leptin as the dependent variable, noninsulindependent diabetes mellitus did not enter the equations at a statistically significant level. The data indicate that there are important gender-based differences in the regulation and action of leptin in humans. Serum leptin levels increase with progressive obesity in both men and women. However, for any given measure of obesity, leptin levels are higher in women than in men, consistent with a state of relative leptin resistance. These findings have important implications regarding differences in body composition in men and women. The observation that serum leptin is not related to energy expenditure rates suggests that leptin regulates body fat predominantly by altering eating behavior rather than calorigenesis. (J Clin Endocrinol Metab 82: 1293 1300, 1997) recombinant leptin (4), suggesting that these mice are defective in the leptin signaling pathway. A high affinity leptin receptor (OB-R) has been identified in the mouse brain (6 8). Studies have linked this gene to the db locus on chromosome 4 in the mouse. Recently, the db mutation has been ascribed to a cytoplasmic domain of the OB-R that is expressed in the hypothalamus as splice variant and is necessary for signal transduction (9). Mutations in the leptin gene have not been linked with obesity in humans (10). However, circulating leptin concentrations are positively correlated with measures of obesity, including body mass index (BMI) and percent body fat (11, 12). In addition, expression of leptin messenger ribonucleic acid (mrna) in adipose tissue is greater in obese than in lean individuals (13). Progressive weight loss during hypocaloric dieting is accompanied by a decline in circulating leptin levels and adipose tissue mrna expression; however, plasma levels then increase once isocaloric diets are initiated to maintain the reduced body weight (12). These observations are consistent with the hypothesis that human obesity 1293

1294 COMMENTS JCE&M 1997 Vol 82 No 4 is characterized by leptin resistance. To understand the basis of hyperleptinemia and leptin resistance, factors that modulate leptin release and mechanisms by which leptin regulates body weight must be identified. For example, it is unclear in humans whether leptin influences energy expenditure or eating behavior as homeostatic effector systems for the maintenance of body weight. Obesity is also associated with diabetes, insulin resistance, and other characteristics of syndrome X (14); however, the relationship of leptin to these metabolic traits has not been studied. We addressed these issues by measuring serum leptin in metabolically well characterized subjects. For the first time, we have demonstrated important gender-based differences in the leptin-obesity axis and that leptin is not likely to influence body fat by modulating resting energy expenditure. Subjects Subjects and Methods The clinical characteristics of the study groups are shown in Table 1. Informed consent was obtained from 85 nondiabetic subjects (50 men and 35 women) and 31 patients with noninsulin-dependent diabetes mellitus (NIDDM; 12 men and 19 women) encompassing a wide range of body weight. Diagnosis of NIDDM was made according to criteria of the National Diabetes Data Group (15), and mean disease duration was 3.5 0.3 yr. Therapy for diabetes included insulin in 4 patients, oral hypoglycemic agents in 21, and diet alone in 6; however, before the study, all NIDDM patients were withdrawn from therapy for at least 3 weeks and followed on an out-patient basis. All subjects studied were chemically euthyroid and without renal, hepatic, or cardiac disease. No subject was ingesting pharmacological agents know to effect carbohydrate homeostasis. In vivo methods In vivo insulin sensitivity was assessed using the hyperinsulinemic euglycemic clamp technique as previously described (16). Briefly, after a 12-h overnight fast, a catheter was inserted into the brachial vein in a antegrade manner to administer infusates. A dorsal hand vein was also cannulated in a retrograde manner and kept in a warming device (65 C) to provide arterialized venous blood for sampling. Regular insulin (Humulin, Eli Lilly Co., Indianapolis, IN) was administrated at a rate of 200 mu/m 2 min through the brachial vein. This infusion rate produced a mean steady state serum insulin concentration of 577 22.2 U/mL, a range of serum insulin that we have found to maximally stimulate glucose uptake and completely suppress hepatic glucose output (17). Blood glucose was clamped at euglycemia (5.0 mmol/l) by measuring glucose in blood taken every 5 min from the dorsal hand vein and adjusting the infusion rate of 20% dextrose solution. To prevent hypokalemia and hypophosphatemia, KPO 4 was infused throughout the study. All studies were performed for at least 3 h, and the glucose infusion rate was constant for the final 60 min of the study. The glucose uptake rate was quantitated in each individual as the mean glucose uptake over the final three 20-min intervals. Whole body glucose uptake was calculated on the basis of the glucose infusion rate corrected for changes in glucose pool size, assuming a distribution volume of 19% body weight and a pool fraction of 0.65. Glucose uptake rates were normalized for lean body mass (excluding bone mass) determined by dual energy x-ray absorptiometry. Mean arterial pressure and heart rate were monitored throughout the study using a Space Labs Recorder (Redmond, WA). Body composition was measured in all subjects by dual energy x-ray absorptiometry scanning (Lunar Radiation, Madison, WI), except that seven subjects were too large or too tall to be accommodated by the scanning device and had body composition determined by underwater weighing. Dual energy x-ray absorptiometry scan measurement of body fat correlates highly with that obtained from underwater weighing (data not shown). Lean body mass determined by these methods reflects muscle mass and was used to normalize glucose uptake data and substrate metabolic rates. Body fat distribution was calculated with waist to hip/thigh measurements. Waist, hip, and thigh circumferences were measured to the nearest 0.5 cm. The waist circumference was taken to the smallest standing horizontal circumference between the ribs and the iliac crest; the hip circumference was taken as the largest standing horizontal circumference of the buttocks, and the thigh circumference was measured at a level 50% of the distance between the knee and the groin. Respiratory calorimetry After an overnight fast, resting metabolic rate was measured over a 30-min period at baseline and at the end of the hyperinsulinemic clamp. Substrate utilization rates were determined by computerized open circuit indirect calorimetry (Deltratrac II, SensorMedics, Yorba Linda, CA). Whole body oxygen consumption (VO 2 ) and CO 2 production (VCO 2 ) were calculated by measuring gradients across the face and the flow rates of air using Haldane transformation. Energy expenditure and rates of lipid and carbohydrate oxidation were determined from the respiratory quotient value and the tables of Lusk (17a). Insulin-induced thermogenesis represents energy expenditure during the final 30 min of the hyperinsulinemic clamp minus the resting metabolic rate. Metabolic rates were normalized per kg metabolically active body mass according to the method of Ravussin et al. (18). Nonoxidative glucose metabolism was calculated by subtracting glucose oxidation rates from total glucose uptake measured during the clamp and was normalized per kg lean body mass. Oral glucose tolerance test and blood pressure Oral glucose tolerance tests were performed as advocated by the National Diabetes Data Group (14). All blood pressure measurements were taken in the dominant arm with the patient having been in the supine position for 30 min. Measurements were made using a calibrated automated cuff device (DynaMap 8100, Johnson and Johnson, New Brunswick, NJ); diastolic and systolic values represent the mean of two readings. Leptin assay Serum for leptin determination was obtained from blood drawn between 0800 0900 h after an overnight fast. Quantification of serum leptin was determined using a RIA and immunopurified rabbit antibodies raised against highly purified recombinant human leptin (Linco, St. Louis, MO). Leptin was labeled with 125 I and purified by gel filtration using Sephadex G-25 (Pharmacia, Piscataway, NJ). For the RIA, dupli- TABLE 1. Clinical characteristics Parameter Normal glucose tolerance Noninsulin-dependent diabetes Male Female Total Male Female Total No. 50 35 85 12 19 31 Age (yr) 34 1.1 36 1.6 35 0.9 48 2.0 45 1.7 45 1.3 Body mass index (kg/m 2 ) 27.9 0.9 25.6 1.0 27.2 0.7 30.2 2.6 31.9 1.2 30.7 1.2 % Body fat 23.9 1.0 32.1 1.6 27.0 1.0 20.3 2.1 34.7 3.0 28.6 2.3 Fasting plasma glucose (mmol/l) 5.1 0.1 4.9 0.1 5.0 0.1 11.1 0.9 12.5 0.8 12.0 0.6 Fasting insulin (pmol/l) 60.9 8.4 38.5 3.6 53.4 5.8 50.3 10.1 89.9 19.4 74.6 12.8 Fasting serum leptin (ng/ml) 6.8 0.6 18.7 2.6 11.6 1.3 6.6 1.4 22.3 2.8 14.7 2.2

COMMENTS 1295 cate 100- L serum samples were added to assay buffer (0.05 mol/l phosphosaline, ph 7.4, containing 25 mmol/l ethylenediamine tetraacetate, 0.1% sodium azide, 0.5% Triton X-100, and 1% RIA grade BSA). Replicate aliquots of quality control pools were included for each assay. After the addition of recombinant leptin and leptin antibody, the samples were vortexed and allowed to incubate at 4 C for 24 h. On the following day, antibody-bound [ 125 I]leptin was precipitated and counted in a -counter. The standard curves were calibrated by the method of Gettys et al. (19), and serum leptin levels were estimated by inverse calibration. The sensitivity of the assay was 0.5 ng/ml, and the concentration of leptin producing 50% displacement from total binding was 5.13 0.23 ng/ml. The interassay coefficient of variation for the quality control values was 6.12%; the intraassay coefficient of variation was less than 5%. Other analyses and statistics Plasma glucose concentrations were measured using the glucose oxidase method and a glucose analyzer (Yellow Springs Instrument Co., Yellow Springs, OH). Fasting serum free insulin levels were measured in all subjects using a double antibody RIA (Abbott Laboratories, Diagnostic Division, Chicago, IL). A fasting lipid profile was assessed after an overnight fast using the Kodak Ektachem Clinical Chemistry Slide (Johnson and Johnson, Rochester, NY). All data analysis was performed with the use of SAS program version 6.10 (SAS Institute, Cary, NC). Data are the mean sem, with sample numbers indicated in the figures and tables. Statistical methods included ANOVA, simple linear regression, and multiple and stepwise regression where indicated. Differences were accepted as significant at P 0.05. Results Gender and the relationship between leptin and obesity To determine the metabolic significance of leptin, 116 subjects were studied over the full range of human obesity (BMI, 17 54 kg/m 2 ; percent body fat, 7 50%) and were characterized on a metabolic ward with respect to obesity, body composition, glucose tolerance, insulin sensitivity, energy expenditure, substrate metabolism, and blood pressure. As shown in Table 1, the study group included 85 individuals with normal glucose tolerance and 31 patients with NIDDM. Serum leptin was measured by RIA in blood obtained between 0800 0900 h after an overnight fast. Figure 1 demonstrates that leptin levels were positively correlated with both BMI (Fig. 1A) and percent body fat (Fig. 1B) in men (r 0.87; P 0.0001 and r 0.82; P 0.0001, respectively) and women (r 0.88; P 0.0001 and r 0.88; P 0.0001, respectively). Importantly, however, we found distinct gender-based differences in the slopes of the regression lines. Leptin levels rose 3.4-fold more rapidly as a function of BMI in women [leptin 1.815 (BMI) 31.103] than in men [leptin 0.534 (BMI) 8.487]. As a result, women exhibited higher leptin concentrations than men (within 95% confidence limits) for any given BMI value above 20 kg/m 2. A similar scenario was observed in the relationship between leptin and percent body fat. Below 25% body fat, women had leptin levels similar to those of men. Above 25% body fat, leptin levels rose 3.2 times more rapidly in women [leptin 1.293 (% body fat) 24.817] than in men [leptin 0.402 (% body fat) 3.087]. Clearly from Fig. 1B, there are women, but no men, with percent body fat values greater than 40%, and there is also a marked preponderance of men with low percent body fat values. As nonoverlapping data points may have unduly influenced the relative slopes of the regression lines, we performed regression analysis of leptin FIG. 1. Relationship between serum leptin and obesity in men and women. Serum leptin levels were measured in 116 subjects (62 men and 54 women) over a wide range of BMI (17 54 kg/m 2 ) and percent body fat (7 50%). In individual men (closed squares) and women (open squares), values for BMI (upper panel) and percent body fat (lower panel) were correlated with serum leptin concentrations. vs. percent body fat for only those men and women with body fat between 29 41%. Over this range there was complete overlap of data points, allowing comparison between genders. Over this delimited range of body fat, leptin levels also rose 3 times more rapidly in women [leptin 1.5562 (% body fat) 35.3971] than in men [leptin 0.4704 (% body fat) 6.4216]. This is important because it illustrates in men and women with similar percent body fat that leptin levels rise more rapidly in women with progressive increments in body fat than in men. Multiple regression analyses substantiated an impact of gender. BMI and gender independently accounted for 35% and 32% of the variability in leptin levels, respectively (both P 0.0001). When percent body fat was taken as the measure of obesity, percent body fat explained 70% and gender explained 7% of the leptin variance (both P 0.0001), and the interaction between these two variables contributed to an additional 4% (P 0.003). Together, percent body fat and gender explained 81% of variance for serum leptin concen-

1296 COMMENTS JCE&M 1997 Vol 82 No 4 trations in humans. Thus, leptin concentrations were higher in women after adjusting for the degree of obesity. Leptin and energy expenditure Leptin can reduce adiposity by both suppressing eating behavior and increasing energy expenditure in mice (1, 4). To assess whether leptin regulates energy expenditure in humans, we measured the resting metabolic rate, which accounts for the major portion of overall energy expenditure (20), in all subjects using indirect calorimetry. As shown in Fig. 2, leptin was not correlated (P NS) with the resting metabolic rate normalized per kg metabolically active body mass in either men or women. In addition, the resting metabolic rate, when included as a covariable with percent body fat or BMI in multiple regression analysis, was not correlated with serum leptin (P NS) in either men or women (not shown). Leptin, diabetes, and syndrome X The presence of overt NIDDM did not affect the relationship between leptin levels and obesity in either gender. Although leptin levels were disproportionately high in women, Fig. 3 demonstrates that mean values of BMI and plasma leptin were similar when comparing nondiabetic and diabetic subgroups in both men and women (P NS). Furthermore, in stepwise and multiple regression models with leptin as the dependent variable and BMI (or percent body fat), gender, and presence/absence of NIDDM as independent variables, NIDDM did not enter the equations at a level even approaching statistical significance. In studies of nondiabetic individuals, we (20a) and others (21, 22) have shown that insulin resistance exists in populations largely independent of obesity. Even so, it is well established that insulin resistance is exacerbated by the development of obesity (23, 24). Therefore, we examined the relationship between leptin and in vivo insulin action by FIG. 2. The relationship between serum leptin and resting metabolic rate. After a 10-h overnight fast, resting metabolic rate was measured by indirect calorimetry and was normalized per kg metabolically active body mass according to the method of Ravussin et al. (17). In individual men (closed squares) and women (open squares), resting metabolic rate was correlated with serum leptin concentrations. FIG. 3. Influence of NIDDM on serum leptin concentrations. The subject groups included 85 nondiabetic subjects (50 men and 35 women) and 31 patients with NIDDM (12 men and 19 women). The figure shows the mean SE values for leptin (white) and BMI (black) in nondiabetic and NIDDM subgroups further subdivided by gender. measuring maximally stimulated rates of glucose uptake during hyperinsulinemic euglycemic clamps. These data are shown in Fig. 4. Interestingly, leptin levels were negatively correlated with maximal glucose uptake rates in men (r 0.57; P 0.0001), whereas these parameters were not clearly related in women (r 0.27; P 0.31). Stepwise regression analysis models were tested with leptin as the dependent variable and percent fat and maximal glucose uptake rate (i.e. insulin sensitivity) as independent covariables. In men, both percent fat (P 0.0001) and maximal glucose uptake rates entered the equation at a statistically significant level. Therefore, insulin sensitivity was related to serum leptin independent of percent body fat in men. In women, only the percent body fat was correlated with leptin; maximal glucose uptake did not enter the regression model at a statistically significant level. During the euglycemic clamp studies, we were able to determine whether hyperinsulinemia could acutely regulate serum leptin levels. In Fig. 5, an approximately 20% increase in leptin concentrations was observed in women; mean leptin concentrations at 120, 150, and 180 min were all significantly greater than the basal level at 0 min (P 0.05). In contrast, leptin levels were not affected (P NS) during identical clamp studies in men. Simple regression analyses relating leptin to traits associated with syndrome X are delineated in Table 2. With respect to fat distribution, leptin levels were highly correlated with upper body obesity, as measured by the waist to hip ratio (r 0.75; P 0.0001) and the waist to thigh ratio (r 0.69; P 0.0006) in men. In women, the correlation between leptin and the waist to hip ratio was less strong (r 0.45; P 0.05) and was not statistically significant compared with the waist to thigh ratio (r 0.10; P 0.68). With regard to glucose tolerance, leptin levels were correlated with fasting blood glucose (r 0.48; P 0.0004 and r 0.36; P 0.03) and with fasting insulin (r 0.51; P 0.0005 and r 0.51; P 0.0004) in both men and women,

COMMENTS 1297 FIG. 5. The effect of induced steady state hyperinsulinemia on serum leptin levels. Euglycemic clamps were performed on 35 subjects. a, Women (n 17; 9 nondiabetic and 8 NIDDM), aged 38.4 2.1 yr; BMI, 28.5 0.9 kg/m 2 ; percent body fat, 33.6 1.9%; maximal glucose disposal rate, 20.3 1.32 mg/kg min in nondiabetics and 10.4 1.6 mg/kg min in diabetics. b, Men (n 18; 13 nondiabetic and 5 NIDDM), aged 36.6 2.4 yr; BMI, 27.1 1.2 kg/m 2 ; percent body fat, 21.4 1.4%; maximal glucose disposal rate, 17.3 1.5 mg/kg min in nondiabetics and 12.6 2.0 mg/kg min in diabetics. Continuous insulin infusion at 200 mu/m 2 min during hyperinsulinemic clamp studies produced a steady state serum insulin level of 577 22 U/mL. This is a marked increase over basal and is sufficient to maximally stimulate peripheral glucose uptake. The figure shows mean serum leptin levels for men (closed squares) and women (open squares) over the 3-h study period. respectively, but were not significantly related to 2-h values of glucose or insulin after an oral glucose challenge (P NS). Therefore, in women, hyperleptinemia is associated with insulin resistance, as reflected by high fasting serum insulin concentrations, but not when quantitated more precisely using the hyperinsulinemia euglycemic clamp technique (Fig. 4). In men, high leptin levels are associated with both elevated fasting insulin and reduced insulin-mediated glucose uptake values. Insulin resistance in syndrome X is also associated with elevated blood pressures (14). Consistent with the strong relationship between leptin and insulin resistance in men, it is not surprising that leptin levels were highly correlated with mean arterial, systolic, and diastolic blood pressures (P 0.0001, P 0.001, and P 0.0007, respectively) in men, but not in women (P NS). However, leptin was not correlated with other parameters that are altered in syndrome X. For example, there was no relationship between leptin and serum triglycerides, high density lipoprotein (HDL) cholesterol, or the triglyceride/hdl ratio in either men or women (P NS). FIG. 4. Relationship between leptin and insulin sensitivity. In vivo insulin action was measured by determining maximally stimulated rates of glucose uptake during hyperinsulinemic euglycemic clamps. Maximal glucose uptake rates were normalized per lean body mass and correlated with leptin levels in men (closed squares, upper panel) and women (open squares, lower panel). Discussion Previous studies have demonstrated that circulating leptin levels in humans are positively correlated with BMI and percent body fat, two measures of obesity (11, 12). As leptin acts to reduce body weight in mice, these data have been interpreted to indicate that overweight humans are characterized by a state of leptin resistance that becomes more pronounced with progressive degrees of obesity. We have confirmed these observations. We also confirm previous data (11, 12) that gender has a major influence on the relationship between serum leptin concentrations and BMI, with leptin levels rising more rapidly as a function of BMI in women than in men. However, at any given BMI, women are likely to have a greater percent body fat than men, making direct body comparisons problematic. We have addressed this issue in the current study. In lean individuals with BMI less than 20 kg/m 2 or percent body fat less than 25%, leptin levels are similar in men and women. With progressive obesity above these values, leptin levels rise 3 times more rapidly in women; consequently, for any measure of BMI or percent body fat, serum leptin levels are significantly higher in women than in men. Although other investigators have confirmed a gender-based difference in leptin levels compared to BMI, other studies have failed to demonstrate a similar

1298 COMMENTS JCE&M 1997 Vol 82 No 4 TABLE 2. Simple regression analyses in nondiabetic individuals Clinical characteristics Pearson coefficient of analysis Nondiabetes males (n 50) Nondiabetic females (n 35) Age 0.30 a 0.13 Obesity Body mass index 0.89 b 0.88 b % Body fat 0.75 b 0.88 b Waist/hip ratio 0.75 b 0.45 a Waist/thigh ratio 0.69 b 0.10 Glucose tolerance Fasting plasma glucose 0.48 b 0.36 a Fasting serum insulin 0.51 b 0.51 c 2-h serum insulin (OGTT) 0.13 0.15 2-h plasma glucose (OGTT) 0.27 0.12 Insulin sensitivity Glucose uptake rate 0.57 b 0.01 Nonoxidative glucose metabolism 0.53 c 0.02 Blood pressure Mean arterial blood pressure 0.59 b 0.24 Diastolic 0.45 c 0.12 Systolic 0.46 b 0.24 Lipid profile Serum cholesterol 0.40 0.46 a Serum triglycerides 0.01 0.24 HDL cholesterol 0.09 0.32 Triglyceride/HDL ratio 0.11 0.32 Energy expenditure Resting metabolic rate 0.01 0.11 Insulin-induced thermogenesis 0.22 0.17 Basal substrate oxidation Respiratory quotient 0.12 0.23 Carbohydrate oxidation 0.13 0.11 Lipid oxidation 0.13 0.05 a P 0.01 0.05. b P 0.001 0.0001. c P 0.01 0.001. gender difference when leptin is compared as a function of percent body fat (11, 12). One possible explanation for discrepant conclusions may involve the range of body weight among study subjects. We demonstrated that only when body fat rises above 25% does gender dissimilarity in leptin levels become apparent. Other studies may not have included an adequate number of sufficiently obese men to resolve the gender effect. Indeed, our data involving percent body fat measurements are consistent with a recent study by Ma et al. (26). It is as yet unclear why there is a gender difference in circulating leptin levels for given measures of generalized obesity. One hypothesis that may explain the higher leptin levels in women involves the role of the sex hormones. It is well known that extremes of body weight are associated with abnormalities in sex hormones and hypothalamic function (25). Marked obesity in women can be associated with dysmenorrhea and infertility, and patients with polycystic ovarian syndrome are characterized by obesity, oligomenorrhea, elevated serum LH/FSH ratio, and hyperandrogenemia (27). On the other hand, patients with anorexia nervosa exhibit suppression of the hypothalamic-pituitary-gonadal axis (28) as well as low levels of serum leptin concentrations, consistent with a reduced amount of adipose tissue mass (Kennedy, A., T. Brewerton, and W. T. Garvey, unpublished data). In remission, these patients gain weight, leptin levels increase, and reproductive endocrine function normalizes. In the current study, we observed higher leptin levels in women over a range of body weight that includes individuals with ideal body weight and modest degrees of obesity. Therefore, it does not appear that abnormalities in sex hormones and hypothalamic function operative at extremes of body weight can explain gender-based differences in leptin. It is more likely that relative leptin resistance in women is due to a physiological effect of sex hormones to modulate leptin at the level of effector systems activated to control body weight. In any event, leptin resistance may provide a partial explanation of why women have a greater percent body fat than men. Predictably, leptin resistance may also make it more difficult for women to sustain diet-induced weight loss. Obviously, these are hypotheses that merit further study. It is important to consider that leptin resistance is a hypothesis based on evidence in rodents that leptin restrains eating behavior and body weight via effects on the hypothalamus. These physiological effects have not been proven in humans. Thus, high circulating leptin in the face of obesity should be considered an apparent state of leptin resistance pending rigorous confirmatory studies in humans. The lipostatic hypothesis, first proposed by Kennedy in 1953 (29), predicted that the body monitors the amount of energy stored in fat and acts to prevent changes from an equilibrium set-point for stored fuel. This theory requires a circulating signal to allow the brain to measure the amount of energy stored in body fat. The adipocyte-derived protein, leptin, is an excellent candidate signal to satisfy this aspect of the hypothesis. However, the mechanism by which leptin regulates body fat has not been fully elucidated. After secretion from adipocytes, leptin appears to interact with specific receptors in the hypothalamus (6 8) and acts to decrease levels of neuropeptide Y (NYP) in the arcuate nucleus and the hypothalamic paraventricular nucleus (30). NPY is implicated in the regulation of energy homeostasis by stimulating food intake and lowering energy expenditure (31, 32). Suppression of NPY by leptin has been hypothesized to activate the sympathetic nervous system and increase energy expenditure via the 3 -adrenergic receptor (33 35) in adipose tissue. If this theory is correct, leptin levels would probably be correlated with energy expenditure rates. The relationship between leptin and energy expenditure has not been studied in humans. We measured the resting metabolic rate, which accounts for the major portion of overall energy expenditure (20, 36), and found no correlation between serum leptin levels and resting metabolic rate in either men or women. This does not exclude a potential influence of leptin on the thermic effect of food or the thermic effect of exercise, which were not measured in this study. However, we also demonstrated that leptin levels were not correlated with insulininduced thermogenesis, a component of overall energy expenditure not reflected in the resting metabolic rate. The data inferentially suggest that leptin may regulate body weight in humans predominantly by affecting behavioral mechanisms and satiety, rather than calorigenesis. We also studied the relationship between serum leptin and metabolic traits associated with syndrome X, including glucose tolerance, insulin sensitivity, and fat distribution. We found that overt diabetes did not alter the relationship be-

COMMENTS 1299 tween leptin and body weight in either men or women, and this is consistent with another recent study (37). Insulin sensitivity was assessed using the hyperinsulinemic clamp technique (37a). Leptin levels were negatively correlated with maximally stimulated glucose uptake rates in men. Thus, serum leptin levels are higher in insulin-resistant men, a finding noted recently (38). In contrast, leptin and maximal glucose uptake rates were not significantly correlated in women. This again highlights gender-based differences in the metabolic significance of leptin. In the male subjects, serum leptin correlated with the pattern of fat distribution, as measured by waist to hip and waist to thigh ratios, whereas these relationships were less pronounced in women. This may be explained by the influence of androgens on fat deposition. Higher androgen levels would consistently determine a pattern of upper body obesity in men, and the gynoid pattern would be more common in women. It is also well known that upper fat distribution is associated with relative insulin resistance (39). In interpreting our data, we favor the hypothesis that leptin regulates overall body fat content, which can then be directed by prevailing androgen levels to an upper body distribution. Relative insulin resistance as a function of truncal fat distribution would then explain its association with higher leptin concentrations in obese men. Body fat could accumulate with a lower or upper body distribution in women, and, therefore, insulin resistance (which is exacerbated only by upper body obesity) would less clearly be related to the hyperleptinemia of obesity. In this view, insulin resistance is not the direct consequence of hyperleptinemia but, rather, the by-product of accumulated truncal fat that occurs in the setting of progressive generalized obesity. Serum leptin was also correlated with elevated blood pressures (diastolic, systolic, and mean arterial pressure) in men, but not in women. These observations can be explained by the facts that leptin is associated with insulin resistance only in men and that insulin resistance is associated with increased blood pressure (14). However, hyperleptinemia was not accompanied by all the characteristics of syndrome X (14). There was no significant correlation with dyslipidemia (triglycerides, HDL, or triglycerides/hdl ratio) in men or women. Also, the relationship between obesity and leptin levels was not affected by the presence of NIDDM. Dyslipidemia and diabetes are components of the ob/ob mouse phenotype and are reversed by exogenous leptin administration. Leptin also influences energy expenditure in mice (1, 4), whereas leptin was not correlated with metabolic rates in humans (resting metabolic rate, insulin-induced thermogenesis, and carbohydrate and lipid oxidation). These combined data indicate that the metabolic effects of leptin may be different in mice and men. The hyperinsulinemic state induced during the clamp led to a significant increase in serum leptin over the basal level in women. This represents an additional gender difference in leptin regulation, because no rise in serum leptin was observed during the 3-h clamp study in men. Insulin-induced hyperleptinemia in women is the first example of acute hormonal or metabolic regulation of leptin demonstrated in humans. It remains possible that, had the hyperinsulinemic clamp been extended for a longer duration, an increase in leptin may have become manifest in men. Other studies (40, 41) have recently assessed the effects of insulin on leptin. Kolacynski et al. (40) reported that leptin levels do not increase acutely with insulin stimulation; however, over an extended time period (i.e. 48 h), hyperinsulinemia led to an increase in circulating leptin levels in both men and women. This study included a preponderance of men over women (69 vs. 8), which could have masked an acute ability of insulin to augment leptin in women but not men. Dagogo-Jack et al. (41) concluded that insulin did not cause an acute increase in leptin on the average; however, inspection of individual curves shows an increase in 25 30% of the subjects. It was not indicated which of these subjects may be male or female. Our data are consistent with studies in mice and rats demonstrating that leptin mrna levels are positively regulated by hyperinsulinemia in adipose tissue (42). In our studies, we have assessed the metabolic significance of morning leptin concentrations. It is now clear that although daytime leptin levels are fairly constant, a large nocturnal rise in serum leptin is observed (43). Therefore, our conclusions pertain only to awake ambient leptin levels. The metabolic importance and influence of gender on the nocturnal leptin increment is unknown. If the nocturnal rise serves only to restrain eating impulses during sleep, then nighttime leptin levels may not have a large impact on the metabolic parameters assessed in our studies. In summary, we have demonstrated a marked influence of gender on the leptin-obesity axis and regulation of serum leptin. Leptin levels increased 3 times more rapidly with progressive obesity in women, as measured by either BMI or percent body fat. Therefore, data suggest that higher levels of leptin are required in women to achieve similar biological end points. This suggests that there may be a difference in the biological homeostatic set-point for obesity in women. In men, leptin levels were highly correlated with measures of insulin resistance and upper body fat distribution, whereas these relationships were less pronounced in women. These latter observations are consistent with the hypothesis that leptin regulates overall body fat mass, which is directed to an upper body distribution by androgens; increasing truncal fat mass can then be more directly implicated in the reduced state of insulin sensitivity. An additional gender-based difference is that induced hyperinsulinemia during euglycemic clamp studies led to an increase in leptin concentrations in women, but not men. Leptin levels were not correlated with resting metabolic rates or insulin-induced thermogenesis in men or women, suggesting that leptin may predominantly regulate adiposity by affecting eating behavior rather than energy expenditure. Additional research is necessary to assess regulation of the leptin-hypothalamic axis by sex steroids and the role of relative leptin resistance in maintaining higher percent body fat in women. Obviously, gender will have to be taken into account when assessing pharmacological interventions using leptin-related drugs to treat human obesity. Acknowledgments We thank Dr. Alan J. Gross, Ph.D., of the Department of Biometry and Epidemiology (Medical University of South Carolina) for assistance with statistics, Ms. Pamela Beasley for typing the manuscript, and John Ber-

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