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Department of Medicine, Division of Endocrinology and Metabolism, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215
Address all correspondence and requests for reprints to: Christian Bjørbæk, 325 Research North, Beth Israel Deaconess Medical Center, 99 Brookline Avenue, Boston, Massachusetts 02215. E-mail: cbjorbae{at}caregroup.harvard.edu
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Introduction |
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Most cases of human obesity, however, are not characterized by reduced levels of functional leptin or by mutations in the gene encoding the leptin receptor. Instead, obesity in both rodents and humans is usually associated with high levels of circulating leptin (12, 13, 14), indicating that subjects develop "resistance" to the anorectic effects of the hormone. Potential mechanisms for leptin resistance include defects in leptin-induced signal transduction in neurons expressing ObRb or defects in transport of leptin into the brain. Consistent with the latter possibility are data showing that obesity in rats and in humans (15, 16) is associated with a decrease in the cerebrospinal fluid (CSF)/serum leptin ratio. Furthermore, some mouse models of obesity that are resistant to peripheral leptin administration respond to leptin delivered directly into the CSF (17, 18).
Leptin has been shown to enter the brain in a specific and saturable manner (19), but the mechanism whereby this occurs is unknown. Brain microvessels and the choroid plexus constitute the blood-brain barrier and blood-CSF barrier (20), respectively, and specific leptin binding has been demonstrated at both sites (6, 21). We recently have shown that purified rat brain microvessels contain high amounts of messenger RNA (mRNA) encoding a short leptin receptor isoform, ObRa (22), which has a predicted 34 amino acid intracellular domain. Collectively, these findings raise the possibility that ObRa is involved in transport of leptin from the circulation into the brain. The purpose of this study was to determine whether ObRa is capable of transcellular transport of leptin in a cell-based model. We stably expressed ObRa in Madin-Darby Canine Kidney (MDCK) cells, a cell type which exhibits polarity when cultured on a substratum (23, 24, 25) and forms functionally distinct plasma membrane domains separated by tight junctions (26). Our findings demonstrate that ObRa is targeted to apical membranes and is capable of unidirectional transport of intact leptin across polarized MDCK cells. These findings support the idea that ObRa may function as a transcellular transporter of leptin from the circulation into the brain at the blood-brain barrier.
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Materials and Methods |
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Stable cell lines
ObRa complementary DNA (cDNA) was cloned as described
previously (27). Due to a low amount of inherent zeomycin
resistance in MDCK cells, ObRa cDNA was subcloned from the original
pcDNA3.1Zeo(-) vector into a pcDNA3.1(-) vector
(Invitrogen, Carlsbad, CA) which conferred neomycin
resistance to stably transfected cells, using standard subcloning
techniques. Transfection of MDCK cells was performed using the
LipofectAMINE system (Life Technologies, Inc.,
Gaithersburg, MD) according to instructions from the manufacturer.
After allowing 48 h for protein expression, cells were split 1:10
or 1:30 into neomycin-containing growth media (Geneticin, 900 µg/ml).
MDCK cells were grown in growth media until colonies of 2- to 3-mm in
diameter were evident. Several independent clones expressing ObRa were
identified by trypsinizing single colonies using cloning cylinders
(Becton-Dickson, Lincoln Park, NJ), followed by individual transfer to
6-well dishes. When a sufficient cell number had been reached, each
clone was assayed for 125I-leptin binding. Stable
MDCK cell lines expressing the Box-1 mutant of ObRa (27) were also
generated.
Cell culture
Stock cells were grown in 10 cm dishes (Becton Dickinson and Co., Lincoln Park, NJ) containing 10 ml of DMEM supplemented
with FCS (final concentration of 10%), 100 U/ml of penicillin and 10
µg/ml of streptomycin at 37 C and 5%CO2. For
both binding and transport studies, cells were plated onto 1
cm2, 0.45 µm polycarbonate filters in sterile
plastic inserts which fit into plastic, premounted transwell system
chambers (Costar, Cambridge, MA). This resulted in an
upper and a lower chamber which allowed access to either the apical or
basolateral surface of the cells. Cell confluence was assessed by
measuring electrical resistance across the cell monolayer with an
Epithelial Voltohmmeter (World Precision Instruments, Sarasota, FL). In
addition, the relationship between electrical resistance and diffusion
of inert 3H-inulin across the cell monolayer was
assessed. Based on results from these studies, binding and transport
assays were performed when a consistent electrical resistance
measurement of more than 150 ohms was obtained, usually on day 4 or 5
after plating of the cells into the transwell system.
Binding assays
To assess ObRa or EGF receptor expression on either the apical
or basolateral cell surface, binding assays were carried out using
either 125I-leptin and ObRa transfected cells or
125I-EGF and parental MDCK cells. Binding of
125I-leptin or 125I-EGF was
carried out at 4 C in transport buffer (HBSS + 0.1% BSA) to minimize
endocytosis and intracellular processing of hormones. Cells were washed
3x with ice cold transport buffer and then incubated with 100,000
cpm/ml 125I-leptin or
125I-EGF added to either the apical chamber (0.5
ml) or basolateral chamber (1.5 ml) for 4 h. To differentiate cpm
bound from nonspecific, background binding, tracer binding was
performed in the presence or absence of excess, unlabeled leptin or EGF
(final concentration of 100 nM). After incubation,
membranes were washed 3x in ice cold transport buffer, excised from
their support with a scalpel and counted using a 
counter. The
number of cpm bound above background was defined as [Total cpm
bound-cpm bound in the presence of excess, unlabeled hormone].
Transport assays
MDCK cells were plated on membranes in triplicate at a starting
confluence of about 30%. After reaching confluence and high electrical
resistance, cells were washed 3x with transport buffer after which
radiolabeled hormone was added to either the apical or the basolateral
chamber. Radiolabeled tracer appearing in the contralateral (CL)
chamber was assessed by collecting 50-µl samples at various time
points and counting in a counter. Sample media was replaced with an
equal volume of fresh media at the time of sample removal. Assays were
carried out at 37 C in 5% CO2. Leptin and EGF
transport were evaluated by adding 100,000 cpm/ml
125I-leptin or 125I-EGF to
either the apical or basolateral chamber in the presence or absence of
excess, unlabeled leptin or EGF. We also used 106
cpm/ml of 3H-inulin to determine the rate of
nonspecific diffusion through the MDCK cell monolayer. Percent diffused
cpm (inulin) or transported cpm (leptin or EGF) was defined as [Total
cpm appearing in CL chamber/cpm radiolabeled ligand added per ml
media] x100.
Degradation analysis
To determine the degree to which transported cpm represented
intact leptin or EGF, trichloroacetic acid (TCA) precipitation was
performed on media collected from chambers contralateral to those to
which radiolabeled ligand was added. Eight hundred microliters of media
were collected from each chamber into 12 x 75 mm polypropylene
tubes. BSA was added to each tube to a final concentration of 0.5%.
800 µl of 10% TCA was added and tubes were incubated on ice for
1 h. This was followed by centrifugation at 4,000x g
for 30 min at 4 C. Because TCA precipitation may result in nonspecific
trapping and coprecipitation, the initial supernatant was collected and
the process repeated on the pelleted fraction with 800 µl of fresh
TCA to give a more accurate assessment of intact leptin. Radioactivity
present in both fractions was counted in a counter. The percent of
intact hormone was calculated as [cpm pellet/combined cpm of both
supernatant fractions + cpm pellet] x 100. Controls consisted of
duplicate samples taken from the radiolabeled hormone stock. To further
affirm that intact leptin was indeed present in the transported media,
proteins in 4 ml of media from the CL chamber were first concentrated
on Centricon concentrators (Amicon, Beverly, MA). The concentrate was
then counted in a counter and proteins were resolved by 15%
SDS-PAGE. An equal number of cpm from the
125I-leptin stock solution were analyzed in
parallel. Gels were then fixed, dried, and finally exposed to a
PhosphorImager cassette (Molecular Dynamics, Inc.,
Sunnyvale, CA).
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Results |
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). At this time, the diffusion rate was
more than 100-fold slower than the rate measured in chambers without
cells (Fig. 1A). The decrease in diffusion after day 3 was correlated
with an increase in electrical resistance between the two chambers
(Fig. 1B). Based on these studies, subsequent leptin and EGF transport
experiments were performed after consistent presence of an electrical
resistance between the two chambers of at least 150 ohm. Similar
electrical measurements have been used by others to determine the
presence of tight junctions between MDCK cells grown in dual-chamber
systems (31, 32).
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, top) occurred primarily in
the apical to basolateral direction and that this transport was
markedly reduced by addition of excess, unlabeled leptin. In contrast,
little radioactivity was detected in the apical chamber following
addition of 125I-leptin to the basolateral
chamber, and these levels were not affected by the addition of excess,
unlabeled leptin suggesting that these low rates likely were due to
passive diffusion.
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, bottom). The percentage
of cpm transported after addition of 125I-leptin
in either the apical to basolateral or basolateral to apical direction
was less than 1.0% after 5 h, which is similar to the nonspecific
diffusion observed for the MDCK-ObRa cells. Furthermore, this amount
was not affected by the addition of excess, unlabeled leptin,
altogether suggesting that no specific transport had occurred. Similar
findings were obtained using cells transfected with empty plasmid
vector (data not shown).
Because MDCK cells exhibit differentiated plasma membranes when grown
on a substratum, we next tested whether the expression of ObRa might be
polarized to either the apical or basolateral cell surfaces (Fig. 3, top). By measuring binding
of 125I-leptin at 4 C to MDCK-ObRa cells, we
found that ObRa was expressed primarily on the surface of the apical
plasma membrane compared with the basolateral side (
8-fold higher).
This result is consistent with the above results showing vectorial
transport of cpm following addition of
125I-leptin in the apical to basolateral
direction. We did not detect any 125I-leptin
binding to either membrane side of parental MDCK cells (Fig. 3, bottom), suggesting that these cells do not express leptin
receptors on either cell surface, consistent with the lack of cpm
transport following addition of 125I-leptin in
nontransfected MDCK cells reported above.
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). Only one band was observed for both
the transport media sample and stock leptin solution and quantitation
by PhosphoImager analysis showed the two bands to be of a similar
density. Since similar cpm of stock 125I-leptin
sample and concentrated transport media sample were loaded onto the
gel, this finding would suggest that the vast majority of radioactivity
in the transport media concentrate represents intact leptin.
Collectively, these results show that although the majority of the
transported cpm represented partially or completely degraded leptin, a
significant fraction (at least 10%) also represented intact leptin,
and demonstrates that ObRa has the capability to mediate transport of
fully intact leptin.
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, top) was approximately 2% after
21 h of incubation, which was reduced to less than 0.5% by
addition of excess, unlabeled leptin. The results for
125I-leptin transport in MDCK-ObRa-Box1 cells
(Fig. 5, bottom) were similar to those obtained for the
MDCK-ObRa cells, i.e. at 21 h of incubation, transport
of leptin was approximately 2% and this was reduced to about 0.6% by
addition of excess, unlabeled leptin. Consistent with these
observations and as shown for wild-type ObRa, we found that ObRa-Box-1
receptors are primarily expressed on the apical cell surface using
125I-leptin binding assays at 4 C (data not
shown). Collectively, these results show that the Box-1 motif is not
required for either proper targeting of ObRa to apical membranes or for
receptor-mediated transport of leptin by ObRa in this system.
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). Consistent with this
finding, there was a 30-fold higher expression of
125I-EGF binding sites on the basolateral
membrane compared with the apical cell surface (Fig. 6B). These results
are consistent with previous results reported by Maratos-Flier and
colleagues (28). Finally, we determined the amount of radioactivity
following transport that represented intact
125I-EGF by TCA-precipitation of media
collected from the apical chamber. We found that intact EGF represented
30.5 ± 2.5% of transported cpm.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Consistent with leptin transport occurring mainly from the apical to basolateral chamber, we found that binding of leptin was about 8-fold higher for the apical cell surface than the basolateral cell surface, suggesting that ObRa is preferentially targeted to the apical membrane in this polarized cell model. Because the basolateral cell surface area of MDCK cells is 4 to 7 times greater than the apical membrane area (34) the concentration of receptors on the apical membrane may actually be 30- to 50-fold higher than that of the basolateral cell membrane. This suggests that the ObRa isoform of the leptin receptor may contain a signal that results in specific sorting to the apical membrane. Similarly, data regarding EGF receptor expression, even after correction for the greater surface area of the apical membrane, indicates that this protein also may contain a sorting signal. However, in contrast to ObRa, this putative signal directs expression of the EGF receptor to the basolateral membrane. Collectively, these data significantly extend our previous observation that ObRa mRNA is abundantly expressed in brain microvessels and choroid plexus (22) and the finding of leptin binding to membranes of isolated human brain microvessels (21). Clearly, further studies are required to directly demonstrate in vivo transport of leptin into the brain via ObRa.
Several studies have shown that proteins expressed in MDCK cells have a similar pattern of polarized expression as seen in vivo (35, 36, 37). Therefore, if ObRa is expressed in apical membranes of brain microvessel endothelial cells, which face the bloodstream, ObRa is well positioned to mediate transport of leptin from the circulation into the brain parenchyma. Leptin would then reach ObRb-containing neurons by diffusion and activate intracellular signal transduction. These findings also suggest that ObRa expression in the choroid plexus, where the apical membrane faces the CSF (20), could serve to mediate leptin clearance from the CSF and the central nervous system. Further studies will be necessary to investigate this possibility.
The endocytic pathway by which the leptin-ObRa complex transverses the MDCK cell monolayer is unknown, but may involve several steps including internalization, degradation, recycling or transcytosis of the ligand and/or the receptor itself. Based on our TCA precipitation data, it appears that the majority of leptin that appears in the basolateral chamber after internalization is partially or completely degraded. This result is consistent with our earlier data showing that ObRa mediates internalization and degradation of leptin via a lysosomal mechanism in CHO cells (38). The fate of receptor bound ligand may depend upon cell type, as suggested by the observation that insulin is largely degraded by adipocytes, but not by endothelial cells (39, 40). Importantly, the present experiments were performed in cells that are derived from the canine kidney. As the kidney serves as a major site of leptin removal and degradation (41, 42, 43) and kidneys express high amounts of ObRa (44), these results suggest that ObRa may play a role in receptor-mediated degradation of leptin at this site in vivo. It might be predicted, then, that a decrease in degradation would result in increased transport of the intact protein. While this is unknown with regard to leptin, in MDCK cells transfected with insulin receptors, inhibiting degradation by the addition of the sulfhydryl alkylating agent N-ethylmaleimide increases the percentage, but not the amount, of intact insulin that is transported (45). We would suggest that leptin may be largely transported as an intact protein across endothelial cells that constitute the blood-brain barrier, while being mostly degraded in the kidney and possibly the choroid plexus. Testing this idea will require assessment of leptin transport in a brain endothelial cell-derived cell line. Nonetheless, a small but significant percentage of leptin was transported in an intact form in our study suggesting that, even in MDCK cells, ObRa possess the capability to mediate transcellular transport of intact leptin.
In summary, we have shown that ObRa is preferentially targeted to the apical cell surface of MDCK cells and can mediate transcellular transport of intact leptin across polarized epithelial cells. These data are supportive of the concept that ObRa mediates passage of leptin across the cerebral microvessels that constitute the blood-brain barrier. Furthermore, deficits in ObRa function at this site could result in leptin resistance. Additional studies are needed to determine whether ObRa function is altered in obesity, which is characterized by peripheral leptin resistance.
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Footnotes |
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Received October 7, 1999.
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stimulates
transcytosis and apical secretion in MDCK cells through cAMP and
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) or on progressive days
following seeding (Day 0) of parental MDCK cells in transwell chambers.
Results for Day 3 (
), Day 4 (
), and Day 6 (•) are shown in
detail in insert. Percent diffused is defined as described in
Materials and Methods. Similar results were obtained
from the B to A direction (not shown). B, Electrical resistance
measured by Epithelial Voltohmmeter on progressive days following
seeding (Day 0) of MDCK cells in transwell chambers. Values represent
the mean ± SD of three measurements.
