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Neuroimaging Research Branch (J.A.M.), National Institute on Drug Abuse, Department of Health and Human Services/National Institutes of Health, Baltimore, Maryland 21224; Neuropsychiatric Institute (E.D.L., B.O.Y., S.C., M.-L.W., J.L.), Department of Molecular and Medical Pharmacology (E.D.L.), David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California 90024; Department of Endocrinology and Metabolism (M.O., S.C.), Gulhane Haydarpasa Training Hospital, Acibadem-Istanbul, 34660 Turkey; and Amgen Inc. (A.M.D.), Thousand Oaks, California 91320
Address all correspondence and requests for reprints to: Edythe D. London, Ph.D., C8-532 NPI, David Geffen School of Medicine, University of California Los Angeles, 760 Westwood Plaza, Los Angeles, California 90024-1759. E-mail: elondon{at}mednet.ucla.edu.
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Abstract |
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Introduction |
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We studied the effects of leptin replacement in the brains of three genetically leptin-deficient adults, the only ones identified so far in the world. Two women and one man, from a highly consanguineous extended Turkish pedigree, were identified with a recessive ob gene mutation (cysteine to threonine in codon 105), homologous to the mutation in ob/ob mice. The resultant leptin deficiency produces multiple hypothalamic abnormalities, punctuated by morbid obesity. Leptin replacement therapy for the three adults consisted of daily sc injections of recombinant methionyl human leptin over the 18 months of study reported here (4).
Structural magnetic resonance (MR) images of the brain were acquired on the three leptin-deficient adults before the initiation of therapy and after 6 and 18 months of leptin replacement. Voxel-based morphometry was used to evaluate possible changes in tissue composition (gray matter concentration) in MR images on a voxel-wise basis (5). In voxel-based morphometry, the concentration of a particular class of tissue (e.g. gray matter) within each voxel is inferred from a weighted average of the intensity values of the neighboring voxels contained within the applied smoothing kernel. Because the range of signal intensities in a brain region on a T1-weighted MR image reflect the relative components of gray matter or white matter, the weighted average intensity in a voxel reflects the tissue concentration of that voxel in the segmented gray matter or white matter image.
Because exogenous leptin administration in ob/ob mice increases brain weight and also increases brain protein and DNA content (6, 7), we hypothesized that analogous treatment of leptin-deficient humans would alter brain tissue composition. Therefore, we tested for increased gray matter tissue concentration from pretreatment levels at 6 and 18 months of leptin replacement therapy.
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Subjects and Methods |
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The three adults (two women, 35 and 40 yr old; and one 27-yr-old man) were recruited in Turkey, and after giving written informed consent, were admitted to the General Clinical Research Center at the University of California Los Angeles for study. There was a baseline adjustment period of 3 months after the subjects arrived in Los Angeles before initiation of leptin replacement therapy. They were allowed to eat ad libitum during their stay, and their level of physical activity was not restricted.
Leptin replacement therapy
A detailed description of the treatment procedure has recently been published (4). The subjects received daily sc injections (each evening between 1800 and 2000 h) of recombinant methionyl human leptin (provided by Amgen, Inc.) at low physiological doses, in the range of 0.01–0.04 mg/kg, for the duration of the study. The dose was recalculated as subjects lost weight, and reduced when normal body mass index was achieved, to avoid excessive weight loss.
MR imaging
Structural MR images were acquired on a Philips Intera 1.5 Tesla scanner at baseline (i.e. before initiation of leptin replacement) and after 6 and 18 months of daily treatment. The parameters for the three-dimensional T1-weighted spoiled gradient-recalled acquisition scan were as follows: TE/TR = 4/24 msec, NEX = 1, flip angle = 35°, field of view = 24 cm, and matrix size = 2562, with 124 sagittal slices of 1.22 mm thickness through the brain.
Voxel-based morphometry
Analysis of the structural MR images was performed using statistical parametric mapping software (SPM99; developed at the Wellcome Department of Cognitive Neurology, University College London). The images were spatially normalized by linear and nonlinear transformations into the standard stereotaxic coordinate space developed at the Montreal Neurological Institute (i.e. MNI coordinate space). The normalized MR images were resampled to an isotropic voxel size of 2 mm. The images were segmented, using a modified mixture cluster algorithm with bias correction for magnetic field inhomogeneity, to produce separate images of gray matter and white matter. The gray matter images were then spatially normalized to the MNI gray matter template images provided in SPM99, to reduce the probability of voxel misclassification. Images from each testing session (baseline, 6 months, and 18 months) were spatially transformed to MNI space independently. The segmented images were finally smoothed with a 12-mm3 isotropic Gaussian kernel to conform the images to the assumptions of random fields theory for statistical analysis (8).
Statistical analysis of MR images
Using the general linear model approach implemented in SPM99, relative differences (changes from baseline MR measurement) in average signal intensity (presumably reflecting changes in tissue composition) were determined within the segmented gray matter volume. All images were proportionally scaled to account for global differences. One-sided contrasts were performed to evaluate increased gray matter tissue concentration (inferred from relative signal intensity) at 6 and at 18 months vs. baseline. The voxel-wise threshold was set at P < 0.0001, and clusters of contiguous voxels that passed this threshold were considered significant at P < 0.001 corrected for multiple comparisons within the entire gray matter volume.
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Results |
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At 6 and 18 months after initiation of replacement therapy, increases in relative gray matter tissue concentration were detected in clusters of contiguous voxels within the frontal cortex, primarily in the left anterior cingulate gyrus, and in the left inferior parietal lobule and the left cerebellum (Table 1
and Fig. 1). No significant effect occurred in the hypothalamic region. The voxels of peak or maximum effect within significant clusters in the left anterior cingulate gyrus (x = –6, y = 32, z = 22), left inferior parietal lobule (x = –46, y = –46, z = 48), and left cerebellum (x = –10, y = –46, z = –46) were at identical stereotaxic coordinates in scans acquired nearly a year apart (i.e. at 6 and 18 months after initiation of leptin replacement). Although the significant clusters were smaller (i.e. fewer voxels) after 18 (vs. 6) months of leptin replacement, the effect in these three regions was sustained (Table 1). All three subjects showed the same direction of effect over the three measurements, i.e. an increase from baseline values in gray matter concentration in the significant voxel clusters.
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Discussion |
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Recent findings suggest that leptin has plastic effects on neural connections between cell groups in the ob/ob mouse hypothalamus (2, 3). We did not detect any increase in tissue concentration in the hypothalamic region after exogenous leptin administration. Our data in only three leptin-deficient adults cannot rule out changes in the hypothalamus but do reveal significant effects on tissue composition in other regions of the brain. In this regard, it is notable that compared with wild-type mice, those that have the ob/ob mutation, and are leptin-deficient, have smaller volumes of many brain areas but not the hypothalamus (9). In addition, the effects of exogenous leptin administration in ob/ob mice, to ameliorate leptin deficiency, were not confined to the hypothalamic region but also affected global measures of brain structure, such as overall brain weight (6). Leptin receptors occur not only in the hypothalamus but also in other brain regions, including the cortex and cerebellum of the human brain (10). In the rat cingulate cortex, labeling of leptin receptors has been detected on neuronal cell bodies (11). Thus, our observations of regional increases in gray matter concentration are consistent with central effects of leptin at leptin receptors.
It is not known to what extent the increases in gray matter concentration reflect local interactions of the hormone with leptin receptors in the brain. Because leptin acts on neurons that coexpress neuropeptide Y, agouti-related protein, proopiomelanocortin, and cocaine- and amphetamine-related transcript (1), our findings could be secondary to the effects of exogenous leptin on one or more of these central-signaling molecules. The localized increases in gray matter concentration also may be secondary to peripheral effects of leptin or other circulating regulatory factors.
The observed effects may reflect increases in neuronal cell volume, reductions in focal myelination, or changes in the neuropil (e.g. dendritic processes). Voxel-based morphometry cannot provide information about the microstructure of a brain region or cytoarchitectonic details. The method can detect a change or a difference in voxel intensity (presumably reflecting tissue concentration), but the mechanism for that effect cannot be determined.
Limitations of the present study need to be recognized. Long-term obesity, as reflected by body mass index, has recently been associated with cerebral atrophy (loss of temporal lobe volume) measured by computed tomography in women (12). Thus, to further establish the validity of the present observations, it will be necessary to include a comparison group of non-leptin-deficient but weight-matched individuals. To our knowledge, voxel-based morphometry has not been used previously to access longitudinal changes in brain tissue composition. However, the identical location of stereotaxic coordinates for the maximum or peak voxel in the significant clusters at 6 months and again at 18 months provides some support for the reliability of the measurement procedure.
The anterior cingulate gyrus, the cerebellum, and the inferior parietal lobule, where we observed increases in gray matter concentration, have all been implicated in neural circuits regulating hunger and satiation in functional imaging studies (13). In this regard, it is interesting that all three of the subjects exhibited dramatic body weight loss although no restrictions were placed on their feeding (4). Nonetheless, the present study can only suggest that leptin may have extrahypothalamic effects on gray matter tissue concentration in the human brain. Although only three genetically leptin-deficient adults could be studied for this preliminary report, it will be important for future studies to extend the present findings, possibly including studies of individuals discovered with other ob gene mutations.
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Footnotes |
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First Published Online February 15, 2005
Abbreviation: MR, Magnetic resonance.
Received October 12, 2004.
Accepted February 4, 2005.
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