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Department of Medicine, Division of Endocrinology (C.B., J.K.L., P.M., R.S.A., J.S.F.), and the Department of Neurology (J.K.E.), Beth Israel Deaconess Medical Center, and Program in Neuroscience (J.K.E.), Harvard Medical School, Boston, Massachusetts 02215; and the Medical Service, Endocrine Division, Department of Veterans Affairs Medical Center and Departments of Medicine and Neurology, Oregon Health Sciences University (A.v.B., A.L.M.), Portland, Oregon 97207
Address all correspondence and requests for reprints to: Dr. Jeffrey S. Flier, Division of Endocrinology, Department of Medicine, Beth Israel Deaconess Medical Center, 99 Brookline Avenue, Research North, Boston, Massachusetts 02215. E-mail: jflier{at}bidmc.harvard.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lack of functional leptin or of long form leptin receptors in ob/ob and db/db mice, respectively, causes severe obesity. Obesity in humans, therefore, may be related to low levels of functional circulating leptin or to decreased action at the target cells expressing long form leptin receptors. Supporting the latter possibility are data demonstrating that serum leptin levels are increased in obesity and correlate positively with body weight (23, 24). This raises the possibility that circulating leptin is not accessible to leptin receptor-expressing neurons in the hypothalamus, or that these cells are themselves deficient in leptin binding or in leptin action. A decreased cerebrospinal-fluid/serum ratio in human obesity has been reported, thus suggesting that a defect in leptin transport into the brain is a possible mechanism for leptin resistance (25, 26).
Very little is known at present about how leptin enters the brain. For leptin to reach most sites within the brain, the hormone must cross the blood-brain barrier (BBB) and/or the blood-cerebrospinal fluid (CSF) barrier. High expression of the short leptin receptor isoform in the choroid plexus (5), the major site for production of CSF, has resulted in speculation that this site is an important site for leptin entry into the brain. On the other hand, consistent with the BBB being a site for transport of leptin into the brain, a recent report demonstrated specific binding of [125I]leptin to isolated human brain microvessels (22). However, whether brain microvessels have the capability to transport leptin has not been demonstrated directly, and the identity of the leptin receptor species on brain microvessels is at present unknown.
Here we applied quantitative RT-PCR in combination with in situ hybridization to identify and quantitate leptin receptor mRNA isoforms in rat brain microvessels. Our results show that the abundance of short form leptin receptor mRNA in brain microvessels is very high, exceeding that of choroid plexus. Other brain regions, including cerebellum, hypothalamus, and meninges express low amounts of this mRNA species. The long form leptin receptor mRNA is present at low levels in the microvessels. In cerebellum, however, the level of long form leptin receptor mRNA is 5 times greater than that in the hypothalamus, 7 times higher than that in microvessels, and 14 times higher than that in the choroid plexus. In situ hybridization revealed strong expression of short leptin receptors in brain microvessels, leptomeninges, and choroid plexus.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-glutamyl
transpeptidase were measured in all microvessel preparations and
compared to original brain homogenates according to the method of
Hausamen et al. (30) and as described previously (29). This
method results in a specific activity of this enzyme in microvessel
samples that is increased approximately 20- to 30-fold compared with
that in the respective original brain homogenates. Total rat brain RNA
was purchased from Ambion (Austin, TX). For in situ
experiments, rats were deeply anesthetized with ip chloral hydrate
(7%; 350 mg/kg) and perfused transcardially with
diethylpyrocarbonate-treated 0.9% saline followed by 500 ml
phosphate-buffered 4% paraformaldehyde (pH 7.0). The brains were
removed, stored in the same fixative for 4 h, and submerged in
20% sucrose in DEPC-PBS, and five series of coronal sections were cut
at 30 µm. The sections were stored at -20 C in an antifreeze
solution until being mounted for in situ hybridization
histochemistry (31).
Quantification of leptin receptor (ObR) mRNA isoforms in rat brain
by RT-PCR
Total RNA from the various tissues was isolated using the
RNA-STAT-60 reagent as described by the manufacturer (Tel-Test,
Friendswood, TX). The complementary DNA (cDNA) was synthesized from 1.0
µg total RNA using deoxythymidine-oligonucleotides and the Advantage
RT-PCR kit from Stratagene (La Jolla, CA). The final volume of the cDNA
samples was 100 µl. For amplification of 764 bp rat ß-actin cDNA,
the following primers were used; upstream primer,
5'-TTGTAACCAACTGGGACGATATGG-3'; and downstream primer,
5'-GATCTT-GATCTTCATGGTGCTAGG-3' (Clontech, Palo Alto, CA). The
following primers were used for specific PCR amplification of 400 bp
(C-terminal 129 amino acids) of rat leptin receptors (ObR) cDNA (short
form): ObR-1, 5'-GTTATATCTGGTTATTGAATGG-3'; and ObR-2,
5'-GAGATACTTCAAAGAGTGTCC-3' (GenBank accession no. D85558). The ObR-2
primer is complementary to the rat ObR short form (ObRs) cDNA in the
region encoding the C-terminus of the receptor and to part of the
3'-untranslated region of the corresponding mRNA (GenBank accession no.
D85557). The following primers were used for specific PCR amplification
of 400 bp (amino acids 930-1063) of rat ObR long form (ObRl) cDNA:
ObR-A,
5-AAAGAGCTCGAGATGGT-ACCAGCAGCTATGG-3';
and ObR-B,
5'-AAAAAGCTTCCCTCCAGTT-CCAAAAG-CTCATCC-3'.
The single underlined sequences are complementary to leptin
receptor cDNA, and the double underlined sequences represent
recognition sites for specific restriction enzymes. These primers were
also used for generation of a probe for in situ
hybridization (see below). Each 50-µl PCR reaction was performed with
5.0 µl cDNA as template. The assay conditions were 10 mM
Tris-HCl (pH 8.8), 50 mM KCl, 1.5 mM
MgCl2, 0.01% gelatin, 0.2 mM deoxy (d)-NTPs,
20 pmol of each primer, 2.5 U Taq polymerase (Stratagene, La
Jolla, CA), and 0.50 µl [
-32P]dCTP (29.6
tetrabecquerels/mmol; 370 megabecquerels/ml; New England Nuclear,
Boston, MA). The mixture was overlaid with 25 µl mineral oil, and
after initial denaturation at 96 C for 3 min, the samples were
subjected to 24–32 cycles of amplification: denaturation at 95 C for 1
min, annealing at 55 C for 1 min, and extension at 72 C for 30 sec.
Five microliters of the reaction were then combined with 5 µl
sequencing stop solution (Amersham International, Aylesbury, UK) and
heated to 85 C for 5 min before loading 4 µl onto a 4%
urea-acrylamide gel (38 x 31 x 0.03 cm). Electrophoresis
was performed at 65 watts of constant power for 3 h before the
gels were transferred to filter paper, dried, and finally subjected to
32P quantification by PhosphorImager analysis (Molecular
Dynamics, Sunnyvale, CA).
Generation of rat leptin receptor complementary RNA (cRNA)
probes
Fragments of rat leptin receptor cDNAs were generated by RT-PCR
using total rat brain RNA (Ambion, Austin, TX) and the Advantage RT-PCR
kit. The following primers were used for amplifying a 500-bp fragment
corresponding to part of the extracellular domain of the leptin
receptor (amino acids 293–460); ObR-C:
5'-AAAGAGCTCACAGCGTG-CTTCCTGGGTCTTC-3'
and ObR-D:
5'-AAAAAGCTTCACAGTGC-TTCCCACTAGTGATTGG-3'.
The single underlined sequences are complementary to rat
leptin receptor cDNA, and the double underlined sequences
represent recognition sites for SacI and
HindIII in primers A and B, respectively. The PCR
products obtained were digested with SacI and
HindIII restriction enzymes and cloned into
pGEM-11Zf+ (Promega, Madison, WI) using standard
techniques. The ObR-A and ObR-B primers from above were used for the
cloning of 400 bp from the ObR cDNA, corresponding to part of the
intracellular domain of the long rat leptin receptor. The inserts of
positive clones were verified by sequencing using standard, double
stranded plasmid techniques. For generation of sense and antisense
35S-labeled cRNA, the plasmids were linearized by digestion
with HindIII or SacI, respectively, and subjected
to in vitro transcription with T7 or SP6 polymerase,
according to the manufacturer’s protocols (Promega).
In situ hybridization histochemistry
The protocol for in situ hybridization histochemistry
was a modification of that previously reported (31, 32, 33). Tissue
sections were mounted onto slides, air-dried, and stored in desiccated
boxes at -20 C. Before hybridization, the slides were immersed in 10%
neutral buffered formalin, incubated in 0.001% proteinase K
(Boehringer Mannheim, Indianapolis, IN) for 30 min and then in 0.025%
acetic anhydride for 10 min, and dehydrated in ascending concentrations
of ethanol. The cRNA probes were then diluted to 106 cpm/ml
in a hybridization solution of 50% formamide, 10 mM
Tris-HCl (pH 8.0), 5 mg transfer RNA, 10 mM dithiothreitol,
10% dextran sulfate, 0.3 M NaCl, 1 mM EDTA (pH
8), and 1 x Denhardt’s solution (Sigma). Hybridization solution
and a glass coverslip were applied to each slide, and sections were
incubated for 12–16 h at 56 C. The coverslips were removed, and the
slides were washed four times with 4 x SSC (standard saline
citrate). Sections were then incubated in 0.002% ribonuclease A
(Boehringer Mannheim) with 0.5 M NaCl, 10 mM
Tris-HCl (pH 8), and 1 mM EDTA for 30 min at 37 C. Sections
were rinsed in decreasing concentrations of SSC containing 0.25%
dithiothreitol: 2 x at 50 C for 1 h, 0.2 x at 55 C for
1 h, and 0.2 x for 1 h at 60 C. Sections were next
dehydrated in graded ethanol (50%, 70%, 80%, and 90%) containing
0.3 M NH4OAc, followed by 100% ethanol. Slides
were air-dried and placed in x-ray film cassettes with BMR-2 film
(Eastman Kodak, Rochester, NY) for 3–5 days. Slides were then dipped
in NTB2 photographic emulsion (Kodak), dried, and stored with desiccant
in foil-wrapped slide boxes at 4 C for 2–3 weeks. Slides were
developed with D-19 developer (Kodak), counterstained with thionin,
dehydrated in graded ethanols, cleared in xylene, and coverslipped with
Permaslip. Sections were analyzed with a Zeiss Axioplan light
microscope using bright- and darkfield optics. Photomicrographs were
produced by capturing images with a digital camera (Kodak, DCS) mounted
directly on the microscope and an Apple Macintosh Power PC computer.
Image-editing software (Adobe Photoshop) was used to combine
photomicrographs into plates, and figures were printed on a dye
sublimation printer (Kodak 8600). Only the sharpness, contrast, and
brightness were adjusted.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-glutamyl transferase and alkaline
phosphatase, respectively, in the microvessel sample vs. the
total brain homogenate, consistent with strong enrichment of cerebral
endothelial cells in the purified microvessel sample (data not shown)
(29). In addition, the preparation was highly enriched in fine
threadlike strands, as determined by microscopy (Fig. 1
). From another set of rats, we isolated
choroid plexi and hypothalami as positive controls for receptor
expression, because these tissues have been shown to express high
amounts of short and long isoforms of leptin receptor, respectively (5, 7). Total RNA was purified in parallel from all tissue samples,
including cerebellum and meninges (mainly dura mater). The total yields
of RNA from microvessels (16 rats), choroid plexi (10 rats), and
meninges (10 rats) were 40, 100, and 75 µg, respectively. The
integrity of the RNA was tested by agarose-gel electrophoresis in
combination with ethidium bromide staining, and no degradation of the
ribosomal RNA bands were detected (data not shown). In addition, the
quality of the RNA from each tissue sample was tested by amplifying
ß-actin cDNA by RT-PCR using limiting cycle numbers, under which
conditions the amounts of ß-actin PCR products were similar (data not
shown).
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). As
shown at the top of Fig. 2, amplification of ObRs from
microvessel and total brain cDNA proceeded at the same rate during the
measured cycles. The two ObRl reactions were also amplified at the same
rate during cycles 24–28, but at a faster rate than the ObRs PCR
reactions (Fig. 2, bottom). However, the cerebellum reaction
entered nonlinear amplification after 28 cycles. We, therefore, chose
26 cycles of amplification for both ObRs and ObRl in the subsequent
quantitative PCR experiments. PCR reactions were then performed in
triplicate for cerebellum, choroid plexus, meninges (dura mater), total
brain, microvessels, and hypothalamus. An autoradiogram of the final
results is shown in Fig. 3. The
quantification of these results by PhosphorImager analysis is shown in
Fig. 4 after normalization to the levels
in hypothalamus. Clearly, the mRNA of the short form of the leptin
receptor is most highly expressed in brain microvessels (Fig. 4, top). This level is even higher than that in the choroid
plexus, the tissue in which ObRs mRNA levels are the highest tested to
date. The mRNA level in the microvessels was more than 40-fold higher
than that in the hypothalamus. The results obtained when amplifying the
long form of the leptin receptor from the same samples are entirely
different (Fig. 4, bottom). Surprisingly, the mRNA levels of
ObRl in cerebellum exceeds those in all other tissues tested, including
the hypothalamus, in which ObRl expression levels were previously
thought to be the highest (7). Low levels of ObRl mRNA were detected in
all other tissues. When no reverse transcriptase enzyme was added to
the samples, no specific RT-PCR product was detected in any sample
(data not shown). Expression of ObRl mRNA was not detected in samples
of peripheral arteries and veins, whereas ObRs mRNA levels were barely
detectable in these tissues (data not shown).
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. Specific
hybridization to perivascular cells from brain capillaries was found in
most regions of the brain, with no particular enrichment in the
hypothalamus compared with the rest of the brain. An example of a
vessel from thalamus is shown in Fig. 5A. No expression of the long
form leptin receptor was detected in microvessels by applying an
antisense probe specific to this isoform (data not shown), thus
supporting our RT-PCR data. Strong specific signals with the all form
probe were also detected in the leptomeninges surrounding the brain
(Fig. 5B), whereas no signal was found in these brain regions when
sections were hybridized with the long form probe (data not shown).
Furthermore, widespread hybridization with the all form probe was
detected in several cell types of the choroid plexus (Fig. 5, C and D).
Very little specific hybridization of the long form probe was seen in
the choroid plexus (data not shown), which agrees with our RT-PCR
results. Both the all form probe and the long form probe showed
extensive hybridization to several hypothalamic nuclei, including the
arcuate nucleus, and to several cellular regions of the cerebellum
(data not shown). In addition, lower intensity specific signals were
found in most parts of the brain with the long form probe (data not
shown). Sense probes for the all form and long form probes did not
exhibit any specific hybridization to any of the above regions of the
rat brain.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The RT-PCR data also show high levels of the mRNA encoding the short isoform of the leptin receptor in the choroid plexus, thus confirming results published previously. The in situ hybridization results indicate that several cell types in the choroid plexus, including epithelial cells and microvessels, express high amounts of ObRs mRNA. These results are consistent with the possibility that the choroid plexus is a site at which leptin is transported into the CSF. Indeed, several studies show that when leptin is administered into the ventricles, it produces similar effects on body weight regulation as leptin injected peripherally (3, 10). When rats received much lower doses of leptin intracerebroventricularly, no effects on food intake were detected (11). The same dose delivered into the hypothalamus did evoke a marked reduction in food intake (11). These results support the possibility that transport of leptin into the CSF via the choroid plexus is not the major route by which leptin reaches the hypothalamus. Furthermore, the concentration of leptin in the CSF is about 0.26 ng/ml in lean humans (25, 26), which is about 40-fold lower than the equilibrium dissociation constant of the leptin receptor (5). Although the latter is based on in vitro results, these data also suggest that leptin is not transported to specific hypothalamic nuclei via the CSF, as the leptin concentration in the CSF is likely to be insufficient to activate significant Janus kinase-STAT signaling by leptin receptors. However, it is at present unknown what role ObRs plays in the choroid plexus and whether the choroid plexus is an important site for transport of leptin into or possibly out of the CSF.
Our in situ data show significant amounts of short form leptin receptor mRNA expression in the leptomeninges of the rat brain. However, the RT-PCR results demonstrated only low receptor mRNA levels in the dura mater of the meninges. Together, these results are consistent with the pia mater and/or the arachnoid being a site of significant expression of ObRs. The function of receptors in the leptomeninges is entirely unknown, and further studies are needed to establish a possible role for ObRs at these sites in the biology of leptin. The possibility that the leptomeninges may degrade CSF leptin should be considered.
The arcuate nucleus of the hypothalamus is a major site of leptin action in the brain, as leptin modulates the expression of neuropeptide Y (10, 35), POMC (13), and AGRP (12) at this site. A recent report demonstrated significant uptake of [125I]leptin into the region of the arcuate nucleus 20 min after iv injection of radioiodinated leptin (21). However, the mechanism by which leptin gained access to this location is not known. As the arcuate nucleus is located near the median eminence, leptin may enter the interstitial fluid by diffusion through the fenestrated capillaries of the median eminence and/or through receptor-mediated transport across the BBB to the arcuate nucleus (21, 34). The Koletsky rat, which lacks all leptin receptor isoforms, has the same level of leptin in the CSF as control rats (36). This suggests that leptin can enter the brain by a mechanism independent of leptin receptors, and one such mechanism might be by diffusion through the circumventricular organs into the CSF. On the other hand, the level of plasma leptin in the Koletsky rat is 10 times higher than in control animals, suggesting saturation of leptin transport into the brain or lack of functional transporters. Careful dose-response and time-course studies of iv injections of gold-labeled leptin or [125I]leptin in combination with electron microscopy may be needed to address these questions.
An unanticipated finding of this study was that the mRNA of the long isoform of the leptin receptor is highly expressed in the cerebellum of the rat. Cerebellar expression exceeds that in other regions of the rat brain including the hypothalamus, the site where leptin is thought to act most potently to regulate body weight. Similar results have recently been reported in a study of the human brain (37, 38). The cerebellum is concerned with the coordination of somatic motor activity, the regulation of muscle tone, and mechanisms that influence and maintain equilibrium. Defects in these functions of the cerebellum have not heretofore been noted in animal models of impaired leptin function or action or in humans with lack of leptin function. Further studies are needed to clarify the biology of these long form leptin receptors in the cerebellum, including the possibility that they bind another, yet unidentified, ligand.
In summary, we have demonstrated that the mRNA encoding the leptin receptor short form is highly expressed in rat brain microvessels. These data support the hypothesis that the BBB is the major site for transport of leptin into the brain. Further studies are clearly needed to characterize this process, including efforts to demonstrate receptor-mediated transcytosis of leptin across brain capillaries. Such studies are critical, given the possibility that the BBB is a major site for leptin resistance in humans.
Received February 3, 1998.
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) and from total brain (
) were subjected
to PCR amplification reactions spiked with [32P]dCTP and
primers specific for the short leptin receptor cDNA. Reactions were
terminated after 24–32 cycles of amplification. Equal amounts from
each reaction were subjected to denaturing urea-PAGE and subsequent
PhosphorImager quantification. Plotted are the average arbitrary units
of measured 32P from duplicate samples.
Bottom, Equal amounts of cDNA derived from 1.0 µg
total RNA from cerebellum (
