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1 From the Massachusetts General Hospital Program in Nutritional Metabolism and the Neuroendocrine Unit, Harvard Medical School, Boston (PK, BC, and SG); the General Clinical Research Center, Massachusetts Institute of Technology, Cambridge, MA (JB); the Department of Pharmacology, University of Virginia Health Sciences Center, Charlottesville, VA (MLJ); and Amgen, Inc, Thousand Oaks, CA (AD).
2 Supported in part by NIH grants M01-RR01066 and M01-RR300088 and by Amgen, Inc. 3 Reprints not available. Address correspondence to SK Grinspoon, Program in Nutritional Metabolism, Massachusetts General Hospital, LON 207, 55 Fruit Street, Boston, MA 02114. E-mail: sgrinspoon{at}partners.
ABSTRACT
Background: HIV-infected patients are affected by changes in fat distribution, ie, significant losses of subcutaneous fat in association with metabolic abnormalities.
Objective: The objective was to investigate the relation between leptin secretion and subcutaneous fat loss in HIV-infected patients.
Design: We investigated leptin pulse dynamics, measured every 20 min overnight from 2000 to 0800 in 41 HIV-infected patients with a mean (±SEM) age of 42.7 ± 1.1 y and body mass index (in kg/m2) of 24.7 ± 0.4 and in 20 healthy control subjects (age: 42.8 ± 1.8 y; body mass index: 24.6 ± 0.5). Leptin pulse variables were compared with total body fat, abdominal subcutaneous fat, and abdominal visceral fat in univariate and multivariate regression analyses.
Results: The number of leptin pulses was not significantly different between the HIV-infected and control subjects. Subcutaneous fat correlated significantly with mean leptin secretion (r = 0.72, P <0.0001), leptin pulse amplitude (r = 0.62, P <0.0001), and leptin nadir (r = 0.62, P <0.0001) in the HIV-infected patients. In stepwise regression modeling, subcutaneous fat (P <0.0001), but not visceral fat, was significantly associated with leptin secretion (overall R2 for the model = 0.57, P <0.0001) in the HIV-infected patients. For each 1-cm2 decrease in abdominal subcutaneous fat area, leptin decreased by 0.044 ng/mL when visceral fat was controlled for. Subcutaneous fat was also significantly related to leptin in the control subjects.
Conclusions: This is the first study to investigate the relation between fat distribution and leptin pulse dynamics in HIV-infected patients. There was a significant reduction in leptin secretion with subcutaneous fat loss in this population.
Key Words: Leptin • pulsatility • subcutaneous fat • HIV
INTRODUCTION
Leptin is a circulating peptide hormone that regulates body weight via its effects on food intake and metabolism. Leptin is secreted primarily from adipocytes, and circulating leptin concentrations are generally proportional to adipocyte mass (1, 2). Previous studies showed that fasting morning circulating leptin concentrations in HIV-infected patients correlate with total body fat (3–5). In addition, Estrada et al (6) showed that plasma leptin concentrations were lower in lipoatrophic patients and correlated with thigh fat. In a recent study of HIV-infected patients receiving antiretroviral medications, a low serum leptin concentration was independently associated with insulin resistance in patients with lipoatrophy, after control for total and regional body fat (7). However, these studies did not assess leptin pulse dynamics or determine the relation of leptin pulse dynamics to specific fat depots in HIV-infected patients with fat redistribution.
In the present study, we investigated leptin pulse dynamics in HIV-infected subjects undergoing blood sampling every 20 min and detailed body-composition assessment. Furthermore, we investigated leptin secretion patterns and pulse dynamics in relation to specific body-composition compartments. In contrast, previous studies have determined the relation between leptin and fat by using a single fasting concentration (3–5), without determination of leptin secretion rates and pulse dynamics.
SUBJECTS AND METHODS
Clinical research protocol
Leptin pulsatility was determined in serum from 41 HIV-infected and 20 control subjects in whom body-composition and metabolic data were described previously (8). Leptin pulsatility was previously analyzed in the control subjects (9). Exclusion criteria included known diabetes mellitus, a hemoglobin concentration <9.0 g/dL, an age of <18 or >60 y, and the use of testosterone, growth hormones, anabolic hormones, glucocorticoids, antidiabetic agents, and any other hormone or medication known to affect neuroendocrine function. Written informed consent was obtained from each subject in accordance with the Committee on the Use of Humans as Experimental Subjects of the Massachusetts Institute of Technology and the Subcommittee on Human Studies at the Massachusetts General Hospital. Subjects were not permitted to eat after 1800 on the night of blood sampling. Samples were collected overnight every 20 min from 2000 to 0800 during an overnight fast. Serum leptin samples were kept refrigerated at –20°F, and all samples were analyzed simultaneously to reduce variability in the technique.
Nutritional assessment and body-composition analysis
Weight was determined after an overnight fast. Percentage ideal body weight was calculated on the basis of data from standard height and weight tables (10). Fat and fat-free mass were determined by dual-energy X-ray absorptiometry (DXA) with a Hologic-4500 densitometer (Hologic Inc, Waltham, MA). The technique has a precision error, in our laboratory, of 1.7% for fat and 2.4% for fat-free mass.
Cross-sectional abdominal computed tomography scanning was performed to assess the distribution of subcutaneous and visceral abdominal fat. A lateral scout image was obtained to identify the concentration of the L4 pedicle, which served as a landmark for the single-slice image. Scan parameters for each image were standardized (144 cm table height, 80 kV, 70 mA, 2 s, 1-cm slice thickness). Fat attenuation coefficients were at –50 Hounsfield Units as described by Borkan et al (11). Abdominal visceral adipose tissue and subcutaneous fat areas were determined by abdominal computed tomography. Food intake was assessed on the basis of a diet history covering the 3 d immediately before the study. Nutrient calculations were performed by using the NUTRITION DATA SYSTEM FOR RESEARCH software version 4.04_31, developed by the Nutrition Coordinating Center, University of Minnesota, Minneapolis, Food and Nutrition Database 31 (released December 2001). The last meal was served to all patients at 1700 and completed by 1800 before the frequent blood sampling began. The calorie, protein, fat, and carbohydrate contents of the final meal were determined for all patients. The last meal before the frequent blood sampling began was ad libitum; however, the calorie, protein, fat, and carbohydrate contents of the meals did not differ significantly between the groups (Table 1).
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TABLE 1. Demographics and leptin pulse characteristics1
Lipodystrophy assessment
The HIV-infected subjects were examined for evidence of increased neck or trunk fat or loss of facial, arm, or leg fat. Fat redistribution was graded on a scale of 0 (none) to 2 (severe) for each of the 5 sites (face, neck, trunk, legs, and arms). Lipodystrophy characterization was determined by a single investigator on the basis of evidence of fat redistribution, including increased fat under the chin, at the back of the neck, in the abdomen, in the chest, and the breast or decreased fat in the arms, legs, or face. The control subjects were healthy and had a waist-to-hip ratio < 0.95 (8). DXA scanning was not used for lipodystrophy categorization.
Hormonal assays
Total human leptin was measured by radioimmunoassay (Linco Research Inc, St Charles, MO) at the Core Laboratory of the Clinical Research Center at the Massachusetts Institute of Technology. For measurement of total human leptin, test plasma (50 µL) was incubated for 24 h at 4°C with assay buffer (containing phosphate-buffered saline and 0.05% Triton X-100), 125I-labeled leptin, and leptin antiserum, and the reagents were incubated for an additional 24 h. Antibody-bound leptin was precipitated with the addition of precipitating reagent. Tubes were centrifuged for 20 min at 3000 x g, after which the supernatant fluid was decanted and the pellets were counted with a counter. To further validate the assay, high and low leptin controls were included in every assay and were required to fall within the manufacturer’s established range for the assay to be accepted. Various concentrations of leptin (4.9–25.6 ng/mL) were added to 5 human serum leptin samples, and the resulting final leptin concentration was measured by the radioimmunoassay. Mean (±SD) leptin concentrations from the 5 separate assays were measured, and the percentage recovery was calculated as the ratio of observed to expected concentrations. Recovery (±SD) was always between 103% and 105% ± 5%. The limit of detection was 0.5 ng/mL. The intraassay CV was <5%, and the interassay CV was <4.5% in our laboratory.
Pulse and deconvolution analysis: pulse and cluster programs
To assess leptin pulsatility we used Cluster, a largely model-free computerized pulse analysis algorithm to identify statistically significant pulses in relation to dose-dependent measurement error in each hormone time series (12, 13). In performing the analysis, we specified individual test cluster sizes for the nadir and the peak width of 2 (2 x 2), a minimum and maximum intraseries CV, a t statistic to identify a significant increase, and a t statistic to define a significant decrease (14). A CV of 5%, the intraassay CV for our assay, was used in the settings of the program. Information about the secretion of the hormone into the serum and the elimination of the hormone from the serum was obtained from PULSE 2 and PULSE 4 (1999 version; University of Virginia, Charlottesville) deconvolution and pulse detection algorithms.
Statistical analysis
Leptin and body-composition variables were compared between all HIV-infected and control subjects by Student’s t test. Leptin pulse variables were compared with body-composition indexes by univariate regression analysis within the HIV-infected group. Multivariate regression modeling was performed for leptin pulse variables, with terms for subcutaneous fat, visceral fat, age, viral load, and medication use. Stepwise regression modeling was performed. Model 1 used P <0.05 to enter, and only subcutaneous fat entered the model. Model 2 used P <0.10 to enter, and both subcutaneous and visceral fat entered the model. With 40 HIV-infected subjects, the study was powered at 0.80 to detect a correlation of 0.43 between endpoints. Statistical analyses were made by using JMP Statistical Database Software (SAS Institute Inc, Cary, NC). Statistical significance was defined as P 0.05. Results are means ± SEMs unless otherwise indicated.
RESULTS
Subject characteristics
Leptin pulsatility was assessed in 41 HIV-infected patients aged 42.7 ± 1.0 y with a BMI of 24.7 ± 0.4 and in 20 simultaneously recruited control subjects aged 42.8 ± 1.8 y with a BMI of 24.6 ± 0.5. Age, BMI, total fat, and food intake (3-d dietary history) were not significantly different between the 2 groups (Table 1). Leptin pulse dynamics were not statistically significantly different between the HIV-infected and control subjects (Table 1). Among the HIV-infected patients, 3.9 ± 0.3 leptin pulses/12 h were observed. Pulsatility varied between 2 and 7 pulses over the 12-h testing period. In the control subjects, 4.7 ± 0.4 leptin pulses/12 h were observed (8). Comparison of the distribution in the number of pulses/12 h between the groups is shown in Figure 1.
FIGURE 1.. Distribution of the number of leptin pulses in the HIV-infected () and the healthy control () subjects determined from frequent (every 20 min) blood sampling from 0800 to 2000.
Relation of leptin pulse dynamics to body fat
Leptin pulse dynamics and mean leptin secretion were highly correlated with total body fat and subcutaneous fat in the HIV-infected subjects (Table 2). Subcutaneous fat correlated highly with peak leptin height (r = 0.62, P <0.0001) and nadir (r = 0.62, P = 0.0001) and overall leptin secretion rates (r = 0.72, P <0.001) (Table 2 and Figure 2). In contrast, the number of leptin peaks did not correlate with body-composition variables in either the HIV-infected or normal control subjects (9). Leptin pulse variables correlated with lipodystrophy scores for extremity fat loss (score for arm fat loss versus leptin secretion rate: r = –0.39, P = 0.03) and truncal fat score (score for truncal fat versus leptin secretion rate: r = 0.37, P <0.05) in the HIV-infected subjects. Univariate and stepwise regression modeling was performed in all of the HIV-infected patients (n = 41; Tables3 and 4). Model 1 (P <0.05 to enter) included only subcutaneous fat and accounted for 52% of the variability in leptin secretion (overall P <0.001). Model 2 (P <0.10 to enter) included both visceral and subcutaneous fat and accounted for 57% of the variability in leptin secretion (P <0.001). Subcutaneous fat was strongly related to overnight leptin concentration and leptin secretion with control for visceral fat, age, and viral load. For each 1-cm2 decrease in subcutaneous fat area, the leptin concentration decreased by 0.044 ng/mL (model 2). Addition of current protease inhibitor use, nucleoside reverse transcriptase inhibitor use, or any antiretroviral use to the model did not change the relation of subcutaneous fat to leptin secretion, and antiretroviral use was not significant in multivariate modeling (data not shown). In the control subjects, leptin was also most highly correlated with subcutaneous fat in both univariate and stepwise regression analyses (Tables 3 and 4 ; 9).
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TABLE 2. Correlation analysis between body-composition indexes and leptin pulse dynamics in HIV-infected patients1
FIGURE 2.. Correlation of mean leptin secretion with total body fat by dual-energy X-ray absorptiometry (r = 0.70, P <0.0001), subcutaneous fat by computed tomography (r = 0.72, P <0.0001), and visceral fat by computed tomography (r = 0.38, P = 0.04) in HIV-infected patients with fat redistribution.
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TABLE 3. Univariate regression model for mean overnight leptin and leptin secretion in HIV-infected patients and healthy control subjects1
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TABLE 4. Stepwise regression models for mean overnight leptin and leptin secretion in HIV-infected patients and healthy control subjects1
DISCUSSION
The concept that adipose tissue is an endocrine organ was strongly supported by the discovery of leptin, which is secreted primarily by adipocytes and acts as a signal to brain regulatory centers controlling energy homeostasis with effects (direct or indirect) on the key organs of metabolism, including the brain, liver, muscle, fat, and pancreas. Plasma leptin concentrations correlate with body fat content (15, 16), but relatively little is known about leptin pulse dynamics in relation to various fat compartments. This issue is particularly relevant for patients with lipodystrophic fat patterns, in whom significant fat loss may occur in the subcutaneous compartment and pulse dynamics are unknown. In addition, patients with lipodystrophic fat patterns may gain visceral fat even as subcutaneous fat is decreased, and the effects of regional fat on leptin pulse dynamics are unknown. Furthermore, because leptin administration has been shown to improve metabolic indexes in such patients and is being contemplated for other lipodystrophic populations, an understanding of baseline leptin physiology in relation to changes in body fat is critical. For example, Oral et al (17) showed that leptin administration improves glycemic control and decreases triacylglycerol concentrations in non-HIV-infected patients with lipodystrophy and leptin deficiency, which suggests that deficiency contributes to insulin resistance and other metabolic abnormalities associated with severe lipodystrophy.
Significant changes in body fat, including loss of subcutaneous fat, increased visceral fat, hepatic steatosis, dorsocervical fat, and lipomatous fat collections as well as increased intramyocellular lipids have been described in HIV patients (18–23). Heterogeneity in fat-distribution patterns has been observed in HIV-infected patients. The etiology of these changes, particularly with respect to highly active antiretroviral therapy, is not known, although recent prospective studies suggest that nucleoside reverse transcriptase inhibitors are associated with subcutaneous fat loss (M Dube, R Zackin, P Tebas, et al, unpublished observations, 2002). The purpose of this study was to characterize the relation of body composition to leptin across a range of fat distribution within a large group of HIV-infected subjects and to investigate the relation of pulse dynamics to regional adiposity. Determination of pulse dynamics from frequent sampling gives more information than does the use of a single static morning sample. In contrast, determination of frequent sampling provides specific information on secretion rates, frequency, pulse height, and area for comparison with detailed measures of body composition.
Our data suggest that there is a significant correlation between subcutaneous fat and leptin pulse secretion in HIV-infected patients with fat redistribution. In the HIV-infected patients, both subcutaneous and visceral fat were significantly related to leptin secretion in univariate regression analysis. In stepwise regression, the subcutaneous fat area remained significantly related to leptin secretion and accounted for a large percentage of the variability in leptin in this model. Our finding that subcutaneous fat is an important source of adipose-derived leptin in HIV-infected patients is consistent with data in non-HIV-infected patients in whom subcutaneous fat is the major fat reservoir (15, 16), constituting on average 80% of the total fat volume in healthy subjects. In contrast, the exact relation between subcutaneous and visceral fat in HIV-infected patients is unknown.
We studied patients with a mixed lipodystrophy phenotype, with subcutaneous fat atrophy and visceral adiposity, enabling us to assess the relation of leptin to opposing changes in regional fat depots simultaneously in one model. Leptin pulse dynamics were not significantly different between HIV-infected and control subjects, because the 2 study groups had similar values for BMI and total body fat. Additional studies of leptin dynamics in HIV-infected patients with pure lipoatrophy, in whom total body and subcutaneous fat are both low, would be of interest. Previous studies have shown that there is no correlation between fasting morning leptin concentration and pulsatility; thus, we investigated leptin pulse dynamics rather than a single leptin measurement (24). Leptin, determined from a single morning fasting concentration, correlates with body fat in HIV-infected patients (3, 4, 6); however, to our knowledge, this is the first study of pulse dynamics with the use of deconvolution techniques in HIV-infected patients with fat redistribution. Leptin pulsatility was previously reported to range from 3.25 pulses/24 h with every 1-h sampling, to 13.4 pulses/24 h with 10-min sampling (25) in non-HIV-infected healthy control and obese subjects. In our study, sampling was performed every 20 min with an algorithm similar to that used in previous studies that investigated leptin pulsatility. For example, Saad et al (26) showed that a 20-min leptin sampling frequency was sufficient to distinguish lean and obese healthy control subjects. Potentially different results might be obtained with a different sampling paradigm. Furthermore, we studied overnight leptin pulsatility, during the time of peak leptin secretion, to assess the relation of body composition to leptin pulse dynamics in this HIV-infected population, independent of the acute effects of calorie intake. No relation was seen between the composition of the last meal or usual calorie intake and leptin secretion patterns. Twenty-four–hour sampling would provide data on the relation of food intake to leptin pulsatility in this population, but that was not the focus of this study.
The metabolism of adipose tissue differs in regional fat depots. Van Harmelen et al (27) showed that subcutaneous fat cells are larger than omental fat cells and that they exhibit increased leptin gene expression. We were not able to assess for regional differences in leptin secretion because we did not collect a biopsy specimen or conduct depot-specific sampling; thus, our results may represent a quantitative effect unrelated to changes in regional fat metabolism. Additional studies are needed to investigate depot-specific regulation of leptin secretion and fat accumulation in HIV-infected patients.
Severe lipodystrophy, caused by a deficiency or destruction of adipose cells, is characterized by low leptin concentrations (28). In the fatless mouse, subcutaneous fat transplantation improves insulin sensitivity and dyslipidemia (29). Gavrilova et al (29) have shown that the improvement in insulin resistance is not due simply to improved glucose uptake by transplanted fat, but also to increased uptake into muscle. Further studies have shown that transplantation of fat lacking leptin does not improve metabolic variables, which suggests that leptin per se is important for restoring metabolic variables in such experiments (30). Leptin administration has been shown to improve insulin resistance in mice and humans with congenital lipodystrophy (17, 31) but has not yet been assessed in HIV-infected patients.
In conclusion, we showed a significant relation between leptin pulse dynamics and subcutaneous fat area in HIV-infected patients with a mixed lipodystrophy pattern. In such patients, leptin secretion, pulse height, and nadir are significantly affected by body composition. In contrast, visceral fat area is less strongly related to leptin pulse dynamics. Further studies of the depot-specific regulation of leptin in HIV-infected patients and of the potential role for leptin in HIV-infected patients with reduced subcutaneous fat and leptin secretion are needed.
ACKNOWLEDGMENTS
We acknowledge the nursing and bionutrition staffs of the General Clinical Research Center of the Massachusetts General Hospital and of the Massachusetts Institute of Technology for their dedicated patient care.
SKG received a research grant from Amgen Inc to conduct this study and contributed to the original design, data collection, and drafting of the manuscript. PK was responsible for the pulsatility analysis and drafting of the manuscript. BC contributed to the pulsatility analysis graphs, figures, and drafting of the manuscript. MLJ provided pulsatility programming support and contributed to the pulsatility analysis. JB contributed to the leptin hormonal analysis by radioimmunoassay. SKG, PK, MLJ, BC, and JB had no financial or personal interest in Amgen Inc. AD is employed by Amgen Inc and contributed to the drafting of the manuscript and provided grant support for the project but was not involved in the data analysis. PK, BC, and SKG performed the data analyses. All authors made critical comments during the preparation of the manuscript and fully accept responsibility for the work.
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