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RECEPTORS |
Department of Physiology, West Virginia University (S.M.H.), Morgantown, West Virginia 26506; Department of Medicine, Division of Endocrinology, Beth Israel Deaconess Medical Center and Harvard Medical School (D.D.P., H.M., C.B., K.E.-H., J.S.F.), Boston, Massachusetts 02215; and Geriatric Research, Education, and Clinical Center, Veteran Affairs Medical Center and Department of Internal Medicine, Division of Geriatrics, St. Louis University School of Medicine (W.A.B.), St. Louis, Missouri 63104
Address all correspondence and requests for reprints to: Dr. Jeffrey S. Flier, Beth Israel Deaconess Medical Center, RN 325, 99 Brookline Avenue, Boston, Massachusetts 02215. E-mail: . jflier{at}caregroup.harvard.edu
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Introduction |
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Several isoforms of Ob-R exist as a result of alternative mRNA splicing (14). One isoform, Ob-Rb, has a cytoplasmic domain containing consensus sequences required for activation of signal transducers and activators of transcription (STATs) and is absent in db/db mice (4, 5, 15). Ob-Rb mRNA has been found at high levels within the hypothalamus and colocalizes with neurons in the arcuate nucleus activated by leptin (16). In addition to Ob-Rb, several short leptin receptors exist. These isoforms exhibit abbreviated intracellular amino acid sequences and have little intracellular signaling capacity (17, 18). Ob-Ra is the most highly characterized short isoform and is expressed at high levels in kidney, lung, and choroid plexus (5, 17). We have recently shown that Ob-Ra mRNA is expressed at very high levels in cerebral microvessels (19), which constitute the blood-brain barrier, raising the possibility that Ob-Ra may play a role in the transport of leptin from the blood into the brain. In support of this idea, leptin binds to human brain microvessel isolates (20), and leptin is taken up into the brain in a specific and saturable manner (21). In addition, we recently showed that Ob-Ra is capable of transporting intact leptin across polarized epithelial cells in vitro (22).
Paradoxically, most cases of obesity are associated with elevated circulating levels of leptin, suggesting that these individuals develop resistance to the anorectic actions of this hormone. Thus, decreased uptake of leptin across the blood-brain barrier may comprise, at least in part, a mechanism for the development of leptin resistance during the evolution of obesity. As several short forms of Ob-R exist, the first objective of this study was to determine the relative expression of the short Ob-R isoforms at the blood-brain barrier. A second aim of these studies was to determine whether Ob-R were required for normal brain uptake of leptin. A third aim was to determine whether leptin uptake into the brain is decreased in rodent models of obesity and whether this might be associated with reduced mRNA expression of short Ob-R isoforms in brain microvessels.
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Materials and Methods |
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Microvessel isolation and quantification of ObR mRNA
Tissues used for quantification of leptin short isoform mRNA levels by RT-PCR were collected in a previous study (19). Briefly, 16 rats were killed, and brains were collected into a buffer containing 118 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 1.0 mM MgSO4, 1.0 mM NaH2PO4, 5.5 mM D-glucose, 0.2% (wt/vol) fraction V albumin, and 28 mM HEPES (pH 7.4). The cerebellum was removed, and the forebrain was stripped of pia mater using a cotton-tipped applicator. As a group, brains were homogenized by hand using a glass-glass Dounce homogenizer (Kontes Co., Vineland, NJ). A series of nylon mesh filtrations was performed to separate microvessels; once through a 149-µm pore size mesh, twice through a 75-µm pore size mesh, and three times through a 37-µm pore size mesh. Microvessels were gently removed from the 75- and 37-µm meshes with a rubber policeman and washed 3 times in PBS. All steps were performed on ice or using sterile, ice-cold solutions. Microvessel isolates were stored at -80 C until RNA was extracted using RNA-STAT 60 reagent as described by the manufacturer (Tel-Test, Friendswood, TX). The cDNA was synthesized from 1.0 µg total RNA as previously described (19). For amplification of rat Ob-R short isoform cDNA, the following primers were used: Ob-Ra (410 bp): upstream, 5'-gattatagtctgttatatctgg-3'; downstream, 5'-gagatacttcaaagagtgtcc-3'; Ob-Rc (405 bp): upstream, 5'-gattatagtctgttatatctgg-3'; downstream, 5'-gggtaatacttaaaaagtgacc-3'; and Ob-Rf (353 bp): upstream, 5'-tatgtcattgtaccgataattatt-3'; downstream, 5'-gggtacctgcacacatatgtg-3'. Each 50-µl PCR reaction was performed with 5.0 µl template cDNA. Assay conditions were 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin, 0.2 mM dNTPs, 20 pmol of each primer, 2.5 U Taq polymerase (Stratagene, La Jolla, CA), and 0.50 µl [
-32P]dCTP (NEN Life Science Products, Boston, MA). The mixture was overlaid with 25 µl mineral oil, and after initial denaturation at 96 C for 3 min, samples were subjected to 25 amplification cycles (which fell within the linear range of amplification for each short Ob-R isoform): denaturation at 95 C for 1 min, annealing at 55 C for 45 sec, and extension at 72 C for 45 sec. Five microliters of reaction product 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, Inc., Sunnyvale, CA).
For quantification of leptin receptor short isoform mRNA levels in various tissues, standard curves were constructed for each isoform. Using 1 µg of either microvessel or choroid plexus RNA, cDNA was synthesized and quantified by spectrophotometry. Values obtained by spectrophotometry were confirmed by agarose gel. The number of molecules per µg cDNA was calculated, then serially diluted to provide 2 x 106, 2 x 105, 2 x 104, and 2 x 103 molecules/µg total RNA standards. Standards and cDNA from the various tissues were then run at the same time in triplicate for the same number of cycles. Values ascertained by PhosphorImager analysis of standards were used to construct a standard curve to which values for tissue PCR products were applied.
Brain leptin uptake in ObRKO mice
To examine the hypothesis that uptake of leptin into the brain is mediated by Ob-R, leptin uptake was determined in mice on a mixed FVB/129/C57BL background which lacked all Ob-R isoforms due to cre-recombinase-mediated Ob-R gene deletion. Both male and female ObRKO mice (n = 10) of various ages were compared with a similar number of age- and sex-matched controls. In most cases, actual littermates were used. Brain uptake of leptin was determined by the brain perfusion method of Banks et al. (23) with seven animals from each group receiving [125I]leptin perfusion and the remaining three mice from each group receiving [125I]leptin in the presence of 5 µM cold leptin. Briefly, mice were deeply anesthetized, and the jugular veins were exposed. An incision was made across the stomach just below the ribs, and the diaphragm was incised. Another cut was made from the base of the sternum to the top of the rib cage. The jugular veins were cut, and the rib cage was spread to expose the heart. The thoracic aorta was clamped, and the perfusion was delivered via the left ventricle of the heart. [125I]Leptin (500,000 cpm/ml; NEN Life Science Products, Boston, MA) was added to infusion buffer, which consisted of 7.19 g/liter NaCl, 0.3 g/liter KCl, 0.28 g/liter CaCl2, 2.1 g/liter NaHCO3, 0.16 g/liter KH2PO4, 0.37 g/liter MgCl2-6H2O, 0.99 g/liter D-glucose, and 10 g/liter BSA (added the day of the infusion). This solution was delivered for 10 min at 1 ml/min, followed by a rapid wash with 20 ml PBS that did not contain radioactivity. Brains were dissected from the cranium, rinsed briefly in PBS, weighed, and then placed in a 12 x 75-mm tube for counting in a 
-counter. The amount of brain uptake of leptin was calculated as [brain cpm leptin/(cpm/µl [125I]leptin infused x brain wt)]. Blood samples were collected from pooled venous blood just before infusion for determination of circulating levels of leptin. To establish the specificity of the method, male C57BL/6J mice received either approximately 500,000 cpm/ml [125I]leptin (n = 5), [125I]leptin, and 1 µM cold leptin (n = 6), or [125I]leptin and 1 µM cold insulin (n = 6).
Brain uptake of leptin and ObR short isoform mRNA levels in isolated microvessels of NZO mice
NZO mice are a polygenic model of obesity originally derived from a pair of agouti mice (24). Due to years of continuos inbreeding, an appropriate lean control strain is not available. Therefore, we used C57BL/6J mice as controls. NZO mice are characterized by resistance to the effect of peripheral, but not central, leptin administration (25) (Hileman, S. M., and J. S. Flier, unpublished data). This suggests that deficient leptin uptake into the brain may comprise a component of the mechanism for obesity in this strain. Brain uptake of leptin for eight NZO and nine C57BL/6J male mice was determined by the brain perfusion method as described above. In this case, however, we added to the perfusate 1,000,000 cpm/ml Tc99m-albumin (as a control for intravascular space and nonspecific uptake). The amount of brain leptin uptake was calculated as [brain cpm leptin/(cpm/µl [125I]leptin infused x brain wt) - brain cpm albumin/(cpm/µl albumin infused x brain wt)]. Uptake of albumin was very low (
10% of leptin values) and did not differ for NZO and control mice. As correction for albumin did not significantly alter the results, this step was omitted from other uptake experiments. Blood samples were collected from pooled venous blood just before infusion for determination of circulating levels of leptin.
To examine mRNA expression for ObR short isoforms in isolated brain microvessels, 11 C57BL/6J and 12 NZO male mice were used. Brain microvessels were collected from each group described above, and leptin receptor short isoforms were assessed by RT-PCR. For each group, 3 separate cDNAs were made, and each were run in triplicate to achieve an adequate estimate of technical error. RT-PCR amplification of product (25 cycles, which fell within the linear range of amplification for each short Ob-R isoform) was performed as described above, but using the following primers: common upstream primer for ObRa and ObRc, 5'-acactgttaatttcacaccagag-3'; downstream ObRa, 5'-agtcattcaaaccattagtttagg-3' (232 bp); downstream ObRc, 5'-tgaacacaacaacataaagcc-3' (260 bp); and ObRall: upstream, 5'-aaagagctcggtcaaaactgctctgcactc-3'; downstream, 5'-aaaaagcttgcagtgacatcagaggtgact. PCR results were normalized to ß-actin mRNA levels using the following primers: upstream, 5'-cgtaccacgggcattgtgatgg-3'; and downstream, 5'-tttgatgtcacgcacgatttccc-3', with reactions ran for 18 cycles.
Effect of a high fat diet on brain uptake of leptin
Our previous work showed that mice made obese by feeding a high fat diet were nonresponsive to peripheral leptin administration, as measured by hypothalamic STAT DNA-binding activity (26). The mice did respond, however, to centrally administered leptin, suggesting the existence of a deficit in brain uptake of leptin. To determine brain uptake of leptin in diet-induced obese mice, 15 male C57BL/6J were fed a diet containing 45% fat for 20 wk. Control mice (n = 10) were fed a diet containing 10% fat. Body weights were measured weekly, and blood was collected just before perfusion to assess circulating concentrations of leptin.
Effect of fasting on brain uptake of leptin and on leptin receptor short isoform mRNA expression in isolated microvessels
A total of 58 age-matched C57BL/6J mice, 12 wk of age, were used. To determine the effect of fasting on brain uptake of leptin, 5 mice were fed ad libitum, 5 mice were fasted for 48 h, and leptin uptake was determined as described above. To examine the effect of fasting on ObR short isoform mRNA levels in brain microvessels, 24 mice were divided equally into 3 groups and fed ad libitum, whereas an additional 24 mice were divided equally into 3 groups and fasted for 48 h. Brain microvessels were collected from each group of 8 animals as described above, and mRNA expression of leptin receptor short isoforms was assessed by RT-PCR as described above.
Internalization of leptin
To determine the amount of brain-associated leptin that internalized vs. that bound to the cell surface, 10 animals were perfused with [125I]leptin as described previously in the absence (n = 5) or presence of 1 µM cold leptin (n = 5), followed by a wash with 20 ml PBS. Another group of 10 animals received similar treatments, except that the PBS wash was replaced with 20 ml of an acidic solution consisting of 0.2 M glacial acetic acid and 0.5 M NaCl (pH 2.0) for the purpose of removing [125I]leptin that might be bound to cell surface receptors. This solution is effective in removing leptin bound to cell surface leptin receptors in vitro (27).
Leptin assays
Samples were collected and placed on ice for about 1 h. They then were centrifuged, and sera was collected and stored at -20 C until assayed. Leptin assays were performed as described previously (12, 28), using a kit provided by Linco Research, Inc. (St. Charles, MO). Samples were run in two assays. The sensitivity of the assays was 0.5 ng/ml, with the intra- and interassay coefficients of variation of 6.0% and 13.0%, respectively.
Statistical analysis
Mean levels of circulating leptin, mean body weights, mean brain weights, brain uptake of leptin, and microvessel ObR mRNA levels for fed and fasted C57BL/6J mice and for NZO mice vs. controls were compared by t test. Results from the ObRKO study, the DIO study, the internalization of leptin study, and the competition of [125I]leptin uptake study were compared by ANOVA, followed by a test of least significant difference (SAS Systems for Windows V8, SAS Institute, Inc., Cary, NC). Due to the limited availability of NZO mice, we were only able to isolate microvessels from one cohort of male NZO mice. To ensure that any possible differences in ObR mRNA levels between control and NZO mice were not due simply to technique-related variation, three separate cDNA preparations were made from the RNA for each group, and each of these were analyzed in triplicate. Means for each cDNA were then derived, and the group means were compared by t test. Differences were considered significant at P < 0.05.
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Results |
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. For all isoforms, serial dilution of standards was accompanied by appropriate decreases in radioactive PCR products. Arbitrary values for standards derived from PhosphorImager analysis were used to construct a standard curve to which values for various tissues were applied, the results of which are shown in Fig. 1B. Ob-Ra mRNA was predominantly found in microvessels and choroid plexus, with lower amounts in the other tissues examined. Ob-Rc mRNA was present in amounts equal to Ob-Ra mRNA in cerebral microvessels, but Ob-Rc mRNA levels were somewhat higher than Ob-Ra in choroid plexus. Relatively high amounts of Ob-Rc mRNA were also observed in cortex and cerebellum, with lower amounts evident in hypothalamus, liver, and lung. Tissue distribution of Ob-Rf mRNA was very similar to that of Ob-Ra mRNA, with levels being greatest in microvessels and choroid plexus. However, in general, Ob-Rf mRNA was present at about 8-fold lower levels than Ob-Ra.
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). In contrast, addition of 1 µM cold insulin did not significantly alter brain uptake of leptin (Fig. 2).
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). Furthermore, uptake in ObRKO mice was not further reduced by the addition of 5 µM excess, cold leptin. This level of uptake was not significantly different from that in control mice receiving 5 µM excess cold leptin.
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), a finding repeated in a subsequent experiment (data not shown) using NZO mice from The Jackson Laboratory. This was not associated with reductions in ObR short isoform mRNA levels, as measured by quantitative RT-PCR (Fig. 4B). Indeed, mRNA expression for ObRa was similar to that in controls, whereas ObRc was elevated. In addition, there was a significant increase in mRNA expression of total Ob-R mRNA.
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). Likewise, circulating levels of leptin were significantly greater for high fat obese animals (33.6 ± 2.1 ng/ml) than for low fat controls (15.6 ± 3.4 ng/ml) or high fat nonobese mice (11.9 ± 2.8 ng/ml), which were not significantly different from each other. Brain uptake of leptin (Fig. 5B) was significantly reduced in obese mice on the high fat diet. Interestingly, uptake was normal in mice fed the high fat diet, but despite this exhibited body weights similar to control-fed mice. We previously reported that expression of Ob-Ra, Ob-Rc, or Ob-Rall mRNA levels in cerebral microvessel isolates was not significantly altered in mice fed a high fat diet (26).
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). Mice used to examine the effect of fasting on ObR short isoform mRNA expression lost a significant amount of body weight (from a prefast weight of 28.6 ± 0.3 to 22.7 ± 0.2 g after 48 h of fasting; P < 0.05) and exhibited a significantly lower level of leptin compared with controls (2.7 ± 0.2 vs. 2.1 ± 0.1 ng/ml; P < 0.05). Despite the increase in brain uptake of leptin, no significant changes in mRNA expression for ObRa, ObRc, or ObRall were observed in response to fasting (Fig. 6B).
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). The combination of 1 µM excess cold leptin and an acidic wash significantly reduced brain leptin uptake, but this inhibition was not different from that observed after adding excess leptin alone. By comparing the degree of reduction observed in animals receiving cold excess leptin alone (i.e. saturable transport) with that observed after washing with an acidic solution, it can be deduced that washing with an acidic solution reduced saturable transport by about 50%.
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Discussion |
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Our study is the first to directly compare, in a quantitative way, Ob-R short isoform mRNA expression levels. Although several studies have reported the tissue distribution of Ob-R, these studies generally have focused on Ob-Ra and Ob-Rb, and due to technical limitations, quantitative comparison of mRNA expression among the various isoforms has not been feasible. As previously shown (19), Ob-Ra mRNA expression was greatest in cerebral microvessels and choroid plexus, structures that constitute the blood-brain and blood-cerebrospinal fluid barriers, respectively. The physiological function of the Ob-Ra short isoform is not clear, but recent evidence suggests that Ob-Ra may function to mediate uptake of leptin from the blood into the brain. Binding of leptin to isolated brain microvessels and saturable, specific uptake of leptin into the brain have been demonstrated (20, 21). Furthermore, we have recently shown that MDCK cells stably transfected with Ob-Ra cDNA are capable of unidirectional, transcellular transport of intact leptin (22).
In contrast to Ob-Ra, almost nothing is known about the possible functions of Ob-Rc or Ob-Rf. In this study we found that Ob-Rc was expressed at equal or greater levels than Ob-Ra in cerebral microvessels and choroid plexus. The existence of Ob-Rc mRNA in areas likely to be important for transfer of leptin into or out of the brain raises the possibility that Ob-Rc could function as a leptin transporter. In addition, and in contrast to Ob-Ra, we found high levels of Ob-Rc mRNA in cortex and cerebellum. Thus, Ob-Rc may have functions different from or in addition to those of Ob-Ra. Although the function of Ob-Rc is unclear, data from our laboratory suggest that CHO cells transiently transfected with murine Ob-Rc DNA are capable of binding, internalizing, and degrading leptin to an extent similar to that of Ob-Ra (27). In contrast to Ob-Ra and Ob-Rc, relatively little mRNA expression for Ob-Rf was noted for any tissue. The relatively low abundance of Ob-Rf mRNA in all tissues examined would suggest that this isoform may be comparatively unimportant or important in only a small subset of cells in the brain. Clearly, more work will be necessary to investigate the functional role of these short Ob-R isoforms. Toward that end, the recent generation of mice lacking all isoforms of Ob-R except for Ob-Rc (31) should be useful in determining the relative role that Ob-Rc may play in leptin transport.
Brain uptake of leptin in the ObRKO mice was significantly decreased. Based on these data, we suggest that a significant portion of brain leptin uptake is mediated by products of the Ob-R gene. This is consistent with the recent finding that brain uptake of leptin is significantly reduced in Koletsky rats, a model that lacks Ob-R (32). Furthermore, it appears that the short isoforms of Ob-R are probably active participants in the transfer of leptin between the blood and the brain. The rate of uptake of leptin by brain is relatively normal in ob/ob mice, which have a full complement of Ob-R but no circulating leptin, and in db/db mice, which express at least the Ob-Ra short isoform, but not Ob-Rb (33). Interestingly, we were not able to further reduce leptin uptake in ObRKO mice, even with a very high level of excess cold leptin. It is possible that this could be related to the brain perfusion technique itself (i.e. the wash step may not be 100% effective). On the other hand, similar to our findings, administration of excess, cold leptin to ob/ob and db/db mice did not completely block brain uptake of leptin (33). This raises two aspects about the nature of leptin uptake by brain: that of the relation between receptor and transporter origins and that of saturable vs. nonsaturable uptake. Although it has been assumed that peptide and protein transporters are simply short forms of receptors, most direct evidence suggests that transporters are separately derived. For example, the transporters for Tyr-macrophage-inhibitory factor-1/Met-enkephalin (34), epidermal growth factor/TGF (35, 36) and IL-1 (37, 38) are all different from the receptors for their ligands. Based on the finding that Ob-R-deficient Koletsky rats had a residual uptake of leptin by brain, Kastin et al. (32) suggested the existence of a transporter that was not one of the known receptors. Such a transporter could be another splice variant of the Ob-R gene, which does not require the posttranscriptional processing deficient in Koletsky rats, or it could be derived from a separate gene. Nonsaturable passage, or transmembrane diffusion, can also be important for peptides (39). Transmembrane diffusion has been described for a number of feeding-related peptides (40, 41, 42, 43, 44). In ICR outbred mice, saturable transport of leptin is about 20 times higher than the predicted nonsaturable component (21). This means that serum leptin levels and adiposity would have to be about 20 times normal to overcome a total lack of transporter. Such levels have been reported, but only in the most obese of individuals. Our acid wash data are consistent these three categories of leptin uptake. The saturable, acid wash-sensitive component corresponds to receptor binding (without internalization/transport), the saturable acid wash-insensitive component corresponds to internalization/transport, and the nonsaturable component, which was independent of vascular space because of the washout step, could be corresponding to transmembrane diffusion. It will be important to determine whether a non-Ob-R-dependent transport system exists for leptin and if it is altered during obesity.
NZO mice and mice made obese by high fat diet ingestion respond to central, but not peripheral, administration of leptin (25, 45, 46). It has been suggested that obesity in both of these models may arise from a decreased blood-brain barrier transport of leptin. In a recent report we observed that mice made obese by a high fat diet did not respond to peripherally administered leptin, as assessed by stimulation of hypothalamic STAT DNA-binding activity. However, they did respond to centrally administered leptin, albeit to a lesser degree than controls (26). This suggests that impaired blood-brain barrier transport of leptin is a component of reduced responsiveness to leptin, and that other deficits may be operative as well. Consistent with this finding, uptake of leptin was reduced in both the NZO mice and high fat diet-induced obese mice. These findings confirm earlier conjecture about reduced brain uptake of leptin in NZO mice and diet- induced obese mice. Also, these findings are consistent with an earlier report that aged, obese CD1 mice exhibit reduced brain leptin uptake (23). Thus, in rodents decreased brain uptake of leptin has been associated with several models of rodent obesity. However, from these studies it could not be determined whether the reduction in brain leptin uptake is causal or merely a result of obesity. Supporting this first possibility is the finding that severely obese ob/ob and db/db mice exhibit normal brain uptake of leptin (33).
In contrast to obesity, fasting for 48 h caused a clear, significant increase in brain uptake of leptin. This would indicate that changes in blood-brain barrier transport of leptin are sensitive to and specific for the physiological imposition that alters circulating concentrations of leptin. This is consistent with the finding of a decreased rate of uptake with escalating, perfused radioactive leptin levels and the suggestion that brain uptake of leptin is most efficient when circulating levels of leptin are low (47). However, these results contrast with those reported by Kastin and Akerstrom (48), which showed that leptin transport significantly declined with prolonged (3–5 d) fasting/starvation. These contrasting results are reconciled to some degree with other findings regarding leptin transport. An increase in exogenous, radioactively labeled leptin transport with short-term fasting is explained by the reduction in competition from endogenous circulating leptin because of the reduction in leptin levels caused by fasting. Later, a decrease in transport with prolonged fasting/starvation should increase the drive to seek food. This explanation assumes that the leptin transporter is itself regulated by factors other than just circulating levels of leptin. Transporter regulation is suggested by the finding that obese ICR mice have an impairment in transporter function not explained by elevated serum leptin levels (23) and that leptin transport can be increased 2- to 3-fold by -adrenergic agonists (49).
Although our findings clearly indicate that brain uptake of leptin is altered during obesity and fasting, the mechanism(s) responsible for this is unclear. As the short Ob-R isoforms seem to be significantly involved in mediating leptin uptake, we originally hypothesized that changes in uptake would be positively associated with changes in mRNA expression for microvessel (e.g. blood-brain barrier) Ob-R short isoforms. We did not find a significant decrease in short Ob-R isoform mRNA expression in association with decreased brain uptake in either NZO mice or diet-induced obese mice. Likewise, fasting for 48 h resulted in increased brain uptake of leptin, but caused no significant changes in short Ob-R mRNA expression in isolated microvessels. Thus, it would appear that changes in brain uptake of leptin are not regulated at the level of mRNA expression for short Ob-R isoforms. Whereas we failed to observe changes in short Ob-R mRNA expression in isolated microvessels, this does not necessarily imply that these systems are unaltered. It is possible that changes in short Ob-R protein expression are occurring without detectable changes in mRNA expression or that another unidentified splice variant is involved. Also, microvessel isolates in these studies represent the brain in its entirety. Therefore, we cannot rule out the possibility that different results might be obtained if either a single vessel type were used or different brain regions had been compared. Binding sites for some cytokines have been largely located to venules (50). As our microvessel preparations represent a collection of several microvessel "types" (i.e. capillaries, arterioles, and venules), potential changes in mRNA levels for short Ob-R isoforms expressed only in a subtype of these vessels potentially would be muted. Also, it is possible that changes in short Ob-R mRNA expression occur in only certain brain regions and cannot be detected in whole brain isolates. That regional variation in microvessel function occurs is suggested by the findings of regional differences in brain uptake of leptin and microvessel protease activity (47, 51, 52). Further investigation will be necessary to determine whether short Ob-R isoform expression and function change in specific types of microvessels and/or in specific regions of the brain during obesity.
In summary, we found that mRNA for both Ob-Ra and Ob-Rc are highly expressed in isolated microvessels; thus, both must be considered when addressing the issue of Ob-R function at the blood-brain barrier. We further showed that Ob-R are an integral part of the uptake process for leptin and that brain uptake was compromised during obesity in NZO mice and mice made obese by ingestion of a high fat diet. Altered uptake was not associated with decreased levels of mRNA for short Ob-R isoforms, suggesting that regulation at this level is not a mechanism by which obesity influences blood-brain barrier function. Thus, it appears that decreased blood-brain barrier transport of leptin comprises at least part of the mechanism of leptin resistance that characterizes most cases of obesity. Development of treatment strategies to circumvent this problem may offer attractive opportunities for pharmaceutical intervention in the pathogenesis of obesity.
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Acknowledgments |
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Footnotes |
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Abbreviations: NZO, New Zealand Obese; Ob-R, leptin receptor; STAT, signal transducer and activator of transcription.
Received July 10, 2001.
Accepted for publication November 5, 2001.
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References |
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, murine IL-1 and murine IL-1ß are transported from blood to brain in the mouse by a shared saturable mechanism. J Pharmacol Exp Ther 259:988–996 binding to mouse brain endothelial cells. J Pharmacol ExpTher 291:665–6701-adrenergic agents. Brain Res 899:209–217[CrossRef][Medline]
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