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Netherlands Institute for Brain Research (U.A.U., J.J.v.H., W.S., D.F.S.), 1105 AZ Amsterdam, The Netherlands; and Departments of Immunology and Rheumatology (J.W.W., E.Z.) and Metabolic Disorders (A.D.H., S.D.F., D.L.H., O.C.P., X.-M.G., D.J.M., L.H.T.V.d.P.), Merck Research Laboratories, Rahway, New Jersey 07065
Address all correspondence and requests for reprints to: D. F. Swaab, Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands. E-mail: dfswaab{at}nih.knaw.nl.
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Introduction |
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One of the orexigenic neuropeptides is melanin-concentrating hormone (MCH) that was first isolated from fish teleost pituitaries in which it is involved in pigmentary control. In the rat, MCH, a cyclic 19-amino acid polypeptide, is synthesized mainly in the lateral hypothalamic area (LHA) and perifornical area but also in a large number of other hypothalamic nuclei, such as the tuberomammillary nucleus (TMN), posterior nucleus, and zona incerta (3, 4). Lactation induces a higher expression of MCH in additional hypothalamic sites such as the medial preoptic nucleus, periventricular nucleus (PeVN), suprachiasmatic nucleus, and paraventricular nucleus (PVN) (5). In humans, the MCH gene is strongly expressed in the hypothalamus (6), and the most prominent cluster of MCH cells runs along the entire rostrocaudal extension of the fornix. MCH nerve fibers are broadly distributed in the rat brain, i.e. in the neocortex, allocortex, hippocampus, basal ganglia, and brain stem (5).
MCH is a leptin-responsive hormone that stimulates feeding, causes obesity (7, 8, 9), regulates the energy balance by acting downstream of leptin (10, 11) and the melanocortin system (12), increases the release of the orexigenic peptides NPY and agouti-related peptide, and decreases the release of the anorexic neuropeptides 
MSH and cocaine- and amphetamine-regulated transcript from hypothalamic explants (13). Central administration of MCH increases food intake in rats (13, 14). In addition, MCH receptor (MCHR) mRNA is up-regulated after food deprivation in the mouse (11, 14). In contrast, mice deficient for MCH have a reduced body weight due to hypophagia and an inappropriately increased metabolic rate, despite their reduced amounts of leptin and proopiomelanocortin mRNA in the arcuate nucleus (12). Continuous MCH1R activation in rat recapitulated the obese phenotype of MCH-overexpressor mice, whereas conversely continuous MCH1R antagonism led to sustained reductions in food intake (15). Furthermore, hypothalamic MCH is induced by cold exposure and participates in the control of energy expenditure in rats (16). In addition, MCH is involved in the regulation of the HPA axis and gonadotropin secretion and the maintenance of the water balance (17), and MCH neurons are strongly active during paradoxical sleep (18).
MCH binds to two G protein-coupled receptors (MCH1R and MCH2R), which are composed of seven-transmembrane domains and regulate several intracellular signaling pathways (19, 20). MCH1R is found in all vertebrates, and MCH1R mRNA expression is regulated by leptin, whereas MCH expression itself has no influence on MCH1R mRNA expression (11). In contrast, MCH2R is absent from rodents but is present in higher mammals including dog, monkey, and man (21). In rodents, MCH1R transcripts are expressed in the arcuate nucleus, in the paraventricular, dorsomedial, and ventromedial nucleus and are up-regulated after fasting (11, 22, 23).
The aim of the present study was to localize the MCH1R in the human hypothalamus and determine the effect of premortem starvation on the expression of MCH1R, in particular in the human infundibular (arcuate) nucleus.
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Materials and Methods |
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New Zealand rabbits were immunized with a 16-amino acid peptide (corresponding to the last 16 residues of human and rat MCH1R: SNAQTADEERTESKGT) conjugated to keyhole limpet hemocyanin (Covance Research, Denver, PA), using procedures detailed previously (24). IgG fractions were prepared from serum by protein A chromatography. IgG from rabbit 1710 (R1710) proved to display the highest titer of 1:150,000 as determined by end point dilution using a solid-phase ELISA and was used for all subsequent experiments.
Validation of R1710 immunolabeling using transiently transfected cell cultures
Human embryonic kidney (HEK)293 cells grown in Falcon 8 chamber culture slides (
80% confluent) were transiently transfected with MCH1R cloned into a PCDNA3 vector. Forty-eight hours post transfection the cultures were fixed and processed for immunolabeling with 5 µg/ml R1710 IgG as described previously (25).
Western blot analysis
Immunoblotting of crude cell membranes from transfected cells was carried out using 35 µg protein and rabbit anti-MHC1R antibody R1710 (10 µg/ml), essentially as described previously (24).
Validation of anti-MCH1R immunolabeling using wild-type and MCH1R knockout rodent brains
Brain tissue was collected from both MCH1R knockout mice [previously described (26)] and their wild-type littermates and also from male adult rats (n = 6 for each) after deep anesthesia and in situ perfusion fixation with 2% freshly prepared paraformaldehyde in 0.1 M phosphate buffer (pH 7.3). After perfusion, the intact brain was removed, fixed for an additional 8–12 h, and then cryoprotected. Then 6-µm cryosections were cut and processed to block nonspecific binding. Preparation of brain tissue and initial handling of tissue sections was carried out as previously described (25). Sections were then incubated with 5 µg/ml R1710 IgG for 60 min at room temperature (RT). Incubation was terminated by extensive washing in buffer. Bound antibodies were detected using the Tyramide signal amplification system (PerkinElmer, Boston MA) with the following modification. Primary antibody was visualized using affinity purified biotinylated F(ab')2 fragments of donkey antirabbit IgG (Jackson Laboratory, Bar Harbor, ME). Digital micrographs were taken through a x63 phase-contrast objective using an Axioplan II microscope (Zeiss, New York, NY). The animal experiment was approved by the institutional animal care and use committee.
Human hypothalamus
Hypothalami of eight cachectic and eight control patients (for clinicopathological data, see Table 1
) were obtained from The Netherlands Brain Bank (coordinator Dr. R. Ravid). Permission was obtained for a brain autopsy and for the use of the tissue and clinical data for research purposes. Tissue blocks were dehydrated in ascending alcohol concentrations, toluene and embedded in paraffin. Subjects were excluded when suffering from: 1) dementia [Braak stage IV-VI (27)]; 2) a primary neurological or psychiatric disease; 3) overweight [i.e. a body mass index (BMI) of > 25 kg/m2]; or 4) when drugs had been taken affecting the hypothalamo-pituitary-thyroid or HPA axis. Additionally, control subjects were excluded when having faced eating problems or loss of weight shortly before death. The criterion for a patient to be cachectic was a BMI of less than 18 kg/m2. But BMI was registered for only five of the 16 subjects. Therefore, some patients were also categorized as cachectic when the medical history showed excessive, well-documented weight loss during the last period of life, often with loss of appetite, vomiting, and/or swallowing problems. The medical records from the control patients confirmed that they were not malnourished (Table 1).
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Serial sections 6 µm thick were cut through the hypothalamus from rostral to caudal. Every 100th section was collected on chromealum gelatin-coated slides and stained with 0.5% thionin for orientation. For the exact localization of the area of the infundibular nucleus, every 100th section of the putative INF was mounted and immunocytochemically stained with a polyclonal NPY antibody (Niepke 26-11-88, Netherlands Institute for Brain Research, antiporcine NPY) (28).
Immunocytochemical localization of MCH1R in the human INF
To localize the MCH1R in the NPY-delineated IFN, every 50th section was mounted on 2% aminoalkylsilane-coated slides. The sections were deparaffinized in xylene and rehydrated in degrading alcohols and distilled water. Slides were washed with Tris-buffered-saline [TBS; 0.05 M Tris, 0.15 M NaCl (pH 7.6); Sigma, Zwijndrecht, The Netherlands] and microwaved in TBS for 10 min at full power (700 W). Sections were cooled down to RT and preincubated with TBS-milk [5% nonfat dry milk in TBS (pH 7.6), Elk, Campina] for 1 h at RT, followed by 1 h incubation (RT) with rabbit IgG, anti-MCH1R 1:500 (R1710) in Supermix-milk [SUMI-milk: 0.25% gelatin (Merck, Amsterdam, The Netherlands) (wt/vol), 0.5% Triton X-100 (Sigma) (vol/vol) in TBS-milk (pH 7.6)] and overnight incubation at 4 C in a moist chamber. The next day slides were washed once with TBS-milk and twice with TBS, followed by incubation in biotinylated goat-antirabbit IgG (Vector Laboratories Inc., Burlingame, CA) 1:400 in SUMI (1 h, RT). Then sections were washed with TBS and incubated with avidin biotin complex (Vector Laboratories) 1:800 in SUMI (1 h, RT) and rinsed in TBS twice. Visualization of the signal was accomplished by incubation in 3.3'-diaminobenzidine (Sigma) 0.5 mg/ml TBS, containing 0.23% (wt/vol) nickelammoniumsulfate (Merck) and 0.01% (vol/vol) H2O2 (Merck) for approximately 20 min (RT). The reaction was stopped in distilled water, and slides were dehydrated in an ascending series of alcohol and xylene and coverslipped in Entellan. Antibody specificity was confirmed by the absence of immunocytochemical staining in the human INF using preimmune serum or antiserum preadsorbed on solid-phase MCH1R peptide (Peninsula Laboratories, Inc., Merseyside, UK; Merck Research Labs; fixed overnight with 4% formaldehyde onto gelatin-coated nitrocellulose filter paper, pore diameter 0.1 µm) (29).
Quantification of MCH1R in the INF
An estimate for the total number of MCH1R-positive neurons in the INF per patient was determined by counting immune-positive cell profiles containing a nucleolus in INF outlines in a set of random systematic selected sections. These measurements were performed using an IBAS-KAT (Kontron KAT-based system) image analysis system. The image analysis system was connected to a SONY black-and-white charge-coupled device camera mounted on a Axioskop microscope (Zeiss). All measurements were performed using a 640-nm small band DEPAL filter (Scott, Mainz, Germany) in the illumination pathway. From the sections to be measured, an image covering the INF (x2.5 objective) was loaded into the IBAS and displayed on the computer monitor. The X-Y scanning stage coordinates of the position of this image in the section were stored. In this image, the INF was outlined manually. For area selection, a grid of rectangular areas superimposed the image, each covering a full-size image on the computer screen at x63 objective. Grid fields in which the outlined INF constituted less than 50% of the field area were excluded. On the basis of the pixel positions of the selected fields in the image and the stored scanning stage position of the x2.5 image, a list of scanning stage coordinates was calculated to put the selected fields in view when looking through a x63 objective. For these calculations the deviations of the optical axis between the objectives were also taken into account.
Next, the x63 objective was positioned in front and under program control the scanning stage moved to the calculated positions. In the image at each position, positively stained profiles containing a nucleolus were counted. All data were stored and an estimate of the total number of MCH1R immunoreactive neurons in the INF was calculated on basis of the profile area, sample size, section thickness, and section number (position) from 11 (± 0.6) different sections from each patient.
Statistics
To examine the effect of cachexia on the number of neurons expressing MCH1R in the human INF/median eminence, the Mann Whitney U test was used. This test was also performed to test for possible differences between the cachectic and control group in postmortem delay, fixation time, or age.
A level of P < 0.05 was used for statistical significance.
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Results |
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To validate the ability of R1710 IgG to immunolabel MCH1R in fixed cells, HEK293 cells were transiently transfected with MCH1R containing vector and subjected to fluorescence immunostaining with R1710 IgG. Fluorescent labeling was observed primarily on the cell membrane of the transfected cells (Fig. 1A) but not in the nontransfected parental cell line (Fig. 1C). This labeling was completely blocked by preincubation with relevant immunizing peptide (Fig. 1B) and was unaffected by incubation with a peptide of similar size but irrelevant amino acid sequence (Fig. 1A). Western blot analysis with R1710 showed an approximately 60-kDa band only in cells transfected with MCH1R but not MCH2R or nontransfected cells (Fig. 1D).
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and 3). Additional, less intensely labeled areas included the caudate putamen, claustrum, periventricular thalamic nucleus, PeVN, habenula, and amygdalohippocampal area (data not shown). In every case R1710 labeling was blocked by preincubation with immunizing peptide. Most importantly, the above-noted structures were labeled only in the wild-type animals but not the MCH1R knockout mice (Fig. 2).
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Although the immunocytochemical staining of neurons was most prominent in the IFN/median eminence (ME) region, MCH1R neuronal staining was also found in the PVN (Fig. 4), LHA, and perifornical area, although in the latter two areas, only few cells were found. In these two latter areas, the PeVN, dorsomedial nucleus (DMN), ventromedial nucleus (VMN), suprachiasmatic nucleus (SCN), and TMN, MCH1R fiber staining was found. Occasionally, nerve terminals in the shape of a basket around a nonstaining neuron were observed in the TMN.
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).
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The general intensity of the immunocytochemical MCH1R staining was higher in the cachectic than the control group (Table 2
and Fig. 5). The mean number of MCH1R cells in the INF/ME of the cachectic group was 1.6 times larger than in the control group (P = 0.02; Fig. 6). There was no significant difference between the two groups for confounding factors as age (P = 0.84), postmortem delay (P = 0.50), or fixation duration (P = 0.70). No difference was observed in the total amount or mean amount of sections used to estimate MCH1R cell number between the groups, nor was there a significant difference between the groups in sex ratio (controls: P = 0.19, cachectics: P = 0.80) or between cancer (n = 6) or noncancer cachexia (n = 2) (P = 0.95), although only a small number of subjects was present in the latter group.
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Discussion |
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There is strong evidence from animal experiments that MCH has a crucial role in the hypothalamic control of feeding and energy regulation (5). The present data would suggest that in humans the response to MCH mainly concerns the cell bodies of the INF/ME area rather than the LHA itself, which contained only few MCH1R-positive cells. Also, the increased number of cell staining for MCH1R in the INF/ME in cachexia agrees with such a function for this brain area. This observed significant increase in MCH1R staining in the human IFN/ME agrees fully with the 7-fold increase in MCH1R mRNA in the mouse brain after 48 h of fasting in mice (11). There were in our series two cachectic subjects with low MCH1R cell numbers [Netherlands Brain Bank (NBB) no. 98-003 and NBB no. 98-117]. These cachectic patients appeared to be tube fed for 1.5 yr (NBB no. 98-003) and 6 wk (NBB no. 98-117), respectively, before their death. The low number of MCH1R-positive neurons indicates a response of MCH1R expression on tube feeding and made it necessary to reanalyze the data without these patients. Omitting these patients and their matched controls leads to an even more significant difference in MCH1R cell number between the control and cachectic groups (P = 0.01).
The presence of MCH1R in not only the IFN/ME but also the PVN, perifornical area, DMN, and LHA may be related to the function of this receptor in mediating orexigenic effects (13, 23). It is currently not known what the chemical nature is of the MCH1R-containing neurons in the PVN, but there are several candidates. Oxytocin (OXT) neurons in the human PVN have a role in food regulation as satiety neurons. It has been shown in animal experiments that OXT neurons projecting from the PVN to brain stem nuclei, such as the nucleus of the solitary tract and dorsal motor nucleus of the vagus, are satiety neurons, and the fact that the number of OXT-containing neurons is decreased in the PVN of Prader-Willi patients is consistent with this possibility (30). Also, CRH neurons in the PVN decrease food intake (31). Double staining of MCH1R and PVN peptides should thus be performed in future experiments. The immunocytochemical staining seen in the PVN and IFN/ME could also be related to the involvement of MCH in the stress response (11, 23). The daily variation of the HPA axis stress response seems to be closely related to food intake as well as basal activity. Also, the MCH1R fiber staining in the SCN, the nucleus that controls circadian rhythms (32), may be related to the stress response because the vasopressin neurons of the SCN inhibit the CRH neurons in the PVN, an effect that contributes also to the hyperactivity of the HPA axis and so to the signs and symptoms of depression (33, 34). The antidepressant and anxiolytic effects of a competitive MCH1R antagonist (SNAP-7941) (35) thus may be very well related to the observed distribution of MCH1R in the PVN and SCN. A role of MCH in paradoxical sleep has been proposed as well (18).
MCH, as well as its receptor MCH1R, are regulated downstream of the leptin-responsive neurons in the arcuate nucleus in rodents. MCH1R regulates feeding and energy balance, i.e. downstream of the melanocortin system (12). Food deprivation decreases leptin levels, which is subsequently followed by an increase in MCH/MCH1R levels. The MCH1R up-regulation, as we observed in cachexia, may therefore be considered, at least partly, as an effect of a decrease in leptin levels. Interestingly, MCH is, moreover, also found in the liver and tongue and may inform the brain as a hormone about the nutritional state of the body in the same manner as leptin (23).
The duration of premorbid illness has previously been correlated with the intensity of NPY and agouti-related peptide staining in the INF/ME (28). It was not possible to match our controls for premorbid illness duration because most people who are ill for a considerable amount of time lose weight. However, no correlation was found between MCH1R cell number count and the duration of premortem weight loss in the cachectic group, which argues against such a confounding effect. A limitation of the present study is the relatively small number of cachectic subjects who were available. The cause of cachexia (cancer vs. noncancer) did not, however, seem to influence MCH1R cell number count, although the number of noncancer cachectic patients was too small to make a solid statement about this important point (n = 5 and n = 2, respectively).
In conclusion, MCH1R staining was found in cell bodies most pronounced in the INF/ME region of the human hypothalamus. The up-regulation of the number of MCH1R-containing neurons in this region in cachexia is consistent with a function of MCH as an orexigenic neuropeptide in the human brain. The presence of MCH1R-expressing neurons and fibers in multiple other sites points to additional functions of the MCH/MCH1R system in humans, including in the stress response and depression, a possibility that warrants further studies.
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Acknowledgments |
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Footnotes |
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First Published Online January 25, 2005
Abbreviations: AVP, Arginine vasopressin; BMI, body mass index; DMN, dorsomedial nucleus; HEK, human embryonic kidney; HPA, hypothalamic-pituitary-adrenal; INF, infundibular nucleus; LHA, lateral hypothalamic area; MCH, melanin-concentrating hormone; MCHR, MCH receptor; ME, median eminence; NPY, neuropeptide Y; OXT, oxytocin; PeVN, periventricular nucleus; PVN, paraventricular nucleus; R1710, rabbit 1710; RT, room temperature; SCN, suprachiasmatic nucleus; TBS, Tris-buffered saline; TMN, tuberomammillary nucleus; VMN, ventromedial nucleus.
Received June 23, 2004.
Accepted January 19, 2005.
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in murine central nervous system: expression in oligodendrocytes and neurons. Brain Res 975:10–21[CrossRef][Medline]
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