Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

The mahogany gene recently has been found to be involved in the suppression of obesity, acting as a signaling receptor (Dinulescu et al., 1998; Nagle et al., 1999). It is expressed in the ventromedial hypothalamic nucleus, the site of action of many ingestive peptides (Nagle et al., 1999). The C terminus of the 1428-amino-acid mahogany protein is a short cytoplasmic tail containing sequences conserved in Caenorhabditis elegans, mouse, and the human being, suggesting a functional domain (Nagle et al., 1999).

The blood-brain barrier (BBB) consists of tight junctions between capillary endothelial cells in the brain that prevent free access of most substances. Although the circumventricular organs lack this form of the BBB, the ependymal cells surrounding these areas are joined by tight junctions to form a second type of restrictive barrier that can provide a less direct form of communication. However, the surface area of the circumventricular organs is at least 5000 times smaller than the high-resistance BBB vessels. This continuous, nonfenestrated endothelial BBB is no longer considered a passive protective barrier. Rather, the BBB can exert a dynamic regulatory function on some peptides and polypeptides (Davson and Segal, 1995; Egleton and Davis, 1997; Strand, 1999).

The most direct and readily regulated form of communication between blood and brain is through saturable (self-inhibitable) transport across the BBB. Although such a saturable blood-to-brain transport system exists for leptin (Banks et al., 1996), it has not been found for most other ingestive peptides (Kastin et al., 1999a). Fortunately, the 1377 to 1428 C-terminal sequence of the mahogany protein contains a tyrosine, suitable for iodination. This facilitated the determination of whether ¹²⁵I-mahogany (1377-1428) crosses the BBB and if so, whether it crosses by a saturable transport system that would enhance its potential for the treatment of obesity.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Adult male albino ICR mice (Charles River, Wilmington, MA), 10 per group with each weighing approximately 22 g, were anesthetized with urethane (4 g/kg, i.p.) and used as approved by the Institutional Animal Care and Use Committee. Mouse mahogany (1377-1428), mol. wt. 5312, was purchased from Phoenix Pharmaceuticals, Inc. (Mountain View, CA). This peptide was radioactively labeled with ¹²⁵I by the chloramine-T method and purified on a column of Sephadex G-10. Acid precipitation showed incorporation of ¹²⁵I into mahogany (1377-1428) of 95%. The specific activity of the ¹²⁵I-mahogany (1377-1428) was approximately 350 Ci/mmol.

Multiple-Time Regression Analysis of Entry into Brain. The ¹²⁵I-mahogany (1377-1428) was injected in a dose of approximately 2.5 pmol/mouse (1.5 × 10⁶ cpm) through the isolated left jugular vein together with 1.5 × 10⁶ cpm of ^99mTc-albumin in 200 µl of lactated Ringer's solution containing 1% BSA. At various times after i.v. injection, blood was collected from a cut in the right carotid artery, and the mouse was immediately decapitated. Serum and brain samples were obtained and counted in a dual-channel gamma counter. The ratio of the radioactivity of brain and serum was calculated, and multiple-time regression analysis was applied to determine the relationship between the ratios and exposure time, the theoretical steady-state integral value of circulating ¹²⁵I-mahogany (1377-1428) after correction for disappearance from blood (Banks and Kastin, 1993).

HPLC in Blood and Brain. Blood and brain samples were obtained 5, 10, 15, and 30 min after i.v. injection of ¹²⁵I-mahogany (1377-1428). The brain was homogenized in PBS with a glass homogenizer to which was added a cocktail of enzyme inhibitors (P8340; Sigma, St. Louis, MO) as well as EDTA and 1,10-phenanthroline to retard degradation induced by the homogenization process. The enzyme inhibitors were not added to the serum samples.

Capillary Depletion with Perfusion. The capillary depletion method was used to separate cerebral capillaries from the brain parenchyma. Each of eight mice received an i.v. injection of ¹²⁵I-mahogany (1377-1428) together with ¹³¹I-BSA in 200 µl of lactated Ringer's solution containing 1% BSA at time 0. At 10 min, four of the mice were perfused intracardially over 30 s with 20 ml of the Ringer's solution while the descending aorta was blocked and bilateral jugular veins were severed. The mice were decapitated, and brain samples were collected. The cerebral cortex was homogenized with a glass homogenizer in physiological buffer and mixed thoroughly with 26% dextran. An aliquot of the homogenate was centrifuged at 5400g in a swinging bucket rotor for 15 min at 4°C.

Efflux from Brain. Approximately 25,000 cpm of both ¹²⁵I-mahogany (1377-1428) and ¹³¹I-BSA were simultaneously injected into the brain of mice anesthetized with urethane at a site 1 mm lateral and 0.2 mm posterior to the bregma through a 1-µl Hamilton syringe (Banks et al., 1997). Mice were studied (n = 6/group) at 0, 2, 5, 10, and 20 min after injection. The 0 min value was determined in mice overdosed with anesthesia before injection, as previously explained (Banks and Kastin, 1989). The half-time disappearance was determined from the regression line obtained from the plot of the logarithm of brain radioactivity against time.

Octanol/Buffer Partition Coefficient. To a mixture of ¹²⁵I-mahogany (1377-1428) and 1 ml of octanol was added 1 ml of a 0.25 M phosphate buffer solution. After being vigorously mixed for 1 min and gently mixed for an additional 10 min, the two phases were separated by centrifugation at 4000g for 10 min. Aliquots were counted for radioactivity, and the partition coefficient was expressed as the ratio of counts per minute in the octanol phase to counts per minute in the buffer phase.

Statistics. Groups were compared by ANOVA followed by Duncan's multiple range test. Regression lines were determined by the least-squares method, and the differences between slopes were compared by GraphPad Prism statistical software (GraphPad, San Diego, CA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Self-Inhibition: Entry of ¹²⁵I-Mahogany (1377-1428) into Brain from Blood after Excess Mahogany (1377-1428). The rate of entry (K_i) of ¹²⁵I-mahogany (1377-1428) into brain was 5.53 × 10⁴ ml/g · min. This value is not corrected for the small influx of albumin, mahogany (1377-1428) fragments, or free ¹²⁵I. The influx of ¹²⁵I, at least, would not be inhibited by the addition of noniodinated mahogany (1377-1428). The decrease of influx (K_i = 1.241 × 10⁴ ml/g · min) after addition of 5.0 µg of unlabeled mahogany (1377-1428) was highly significant (P = .001). The excess mahogany (1377-1428) decreased the entry of ¹²⁵I-mahogany (1377-1428) to a level not significantly different from that of either ^99mTc-albumin alone or ^99mTc-albumin with the unlabeled mahogany (1377-1428). The K_i of ^99mTc-albumin was not significantly different from zero in mice injected with ¹²⁵I-mahogany (1377-1428) or with the unlabeled mahogany (1377-1428), indicating a lack of disruption of the BBB. The results are shown in Fig. 1.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Self-inhibition of influx: blood-to-brain entry of 125I-mahogany (1377-1428) (I-mahogany) with and without the addition of 5 µg/mouse unlabeled mahogany (1377-1428). The entry of 125I-mahogany (1377-1428) was much faster than that of 99mTc-albumin, the vascular control, and was significantly inhibited by excess mahogany (1377-1428), indicating the self-inhibition characteristic of a saturable transport system (n = 8-14 mice/group).

Cross-Inhibition: Entry of ¹²⁵I-Mahogany (1377-1428) into Brain after Excess -MSH, Leptin, AgRP (83-132), MCH, EGF, and JKC-363. None of these related peptides significantly reduced the K_i of ¹²⁵I-mahogany (1377-1428) or changed the K_i of ^99mTc-albumin when added to the injectate at a dose of 5 µg/mouse. Nevertheless, in each of the three experiments in which the ability of -MSH to inhibit the transport of ¹²⁵I-mahogany (1377-1428) was tested, the addition of -MSH resulted in a slightly lower K_i for ¹²⁵I-mahogany (1377-1428).

HPLC. In serum, the radioactivity eluting at the same position as the ¹²⁵I-mahogany (1377-1428) standard, corrected for processing, showed 75.0% intact at 5 min, 75.3% intact at 10 min, 51.0% intact at 15 min, and 44.2% intact at 30 min. In brain, 66.3% was intact at 5 min, 64.2% at 10 min, and approximately 40% at the later times. The calculated half-time disappearance of the intact ¹²⁵I-mahogany (1377-1428) in serum was 25.8 min and in brain was 25.5 min. These results are shown in Fig. 2.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   HPLC: disappearance from blood of intact 125I-mahogany (1377-1428) after i.v. injection. The top line and closed circles represent serum; the bottom line and open circles represent brain. The half-time disappearance from both blood and brain was approximately 26 min (n = 5 mice).

Capillary Depletion. Ten minutes after i.v. injection of ¹²⁵I-mahogany (1377-1428) there was nearly 50 times more radioactivity in the parenchyma than was bound to the capillaries (P < .00001). Significantly (P < .0001) more ¹²⁵I-mahogany (1377-1428) was reversibly associated with the vasculature than was tightly bound to the capillaries. These results are shown in Fig. 3.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Capillary depletion: tissue to serum ratios for mice injected with 125I-mahogany (1377-1428) corrected for simultaneously injected 131I-albumin. Values for the cortex are composed of three components: parenchyma (remaining after washout), capillaries (remaining after washout), and reversible vascular association (removed by washout). Significantly more 125I-mahogany (1377-1428) was found in the parenchyma (and washout) than was bound to the capillaries (n = 4 mice/group).

Efflux from Brain. The half-time disappearance from brain was 29.0 min for ¹²⁵I-mahogany (1377-1428) and 11.4 min for ¹³¹I-BSA. This difference was highly significant (P < .001). Because the half-time disappearance of ¹²⁵I-mahogany (1377-1428) was much slower than that of ¹³¹I-BSA, there was no brain-to-blood transport system for ¹²⁵I-mahogany (1377-1428). It left the brain at a rate slower than the usual reabsorption of cerebrospinal fluid. The results are shown in Fig. 4.

View larger version (10K): [in this window] [in a new window]   Fig. 4.   Efflux: disappearance of 125I-mahogany (1377-1428) (I-mahogany, ) and 131I-albumin (albumin, ) from brain after i.c.v. injection. The rate of disappearance of 125I-mahogany (1377-1428) was less than that of the 131I-albumin, indicating retention rather than efflux (n = 5-7 mice/group).

Octanol/Buffer Partition Coefficient. The octanol coefficient, calculated as the counts per minute in the octanol phase divided by the counts per minute in the buffered saline phase was 0.0050 ± 0.0004 for ¹²⁵I-mahogany (1377-1428).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The MC₄ receptor, important in the regulation of feeding behavior, is activated by -MSH and antagonized by AgRP (83-132) (Dinulescu et al., 1998; Rossi et al., 1998; Ollmann et al., 1999). It has been proposed that the mahogany protein could act by interference with AgRP processing or secretion or by disruption of its desensitization of melanocortin receptors (Dinulescu et al., 1998; Gunn et al., 1999). The mahogany locus, when homozygous, blocks the obesity resulting from ectopic expression of the agouti locus in brain even though leptin concentrations are unaffected (Dinulescu et al., 1998). Mahogany is required for the actions of AgRP, but its effects on the stimulation of feeding are independent of its suppression of the agouti locus (Dinulescu et al., 1998) or the MC₄ receptor (Nagle et al., 1999).

The mahogany locus does not affect circulating concentrations of -MSH (Miller et al., 1997), and we found that the slight inhibitory effect of -MSH on entry of ¹²⁵I-mahogany (1377-1428) was not statistically significant. In addition, AgRP (83-132), MCH, and an MC₄ receptor antagonist were not able to inhibit the entry of mahogany (1377-1428) into brain. Furthermore, even though the mahogany protein contains three EGF domains (Nagle et al., 1999), EGF did not inhibit its entry. Leptin, also, did not show cross-inhibition of ¹²⁵I-mahogany (1377-1428).

Measurement of radioactivity in brain after peripheral administration could be misleading if there were an active efflux system from brain to blood such as exists for corticotropin-releasing hormone (Martins et al., 1996, 1997). Not only did we find no evidence for an active egress process for ¹²⁵I-mahogany (1377-1428), but the results indicate that this peptide was retained in the brain, perhaps related to receptor association or cellular uptake.

Other possibilities that could misleadingly affect interpretation of the influx results also were eliminated. HPLC showed that most of the mahogany (1377-1428) reaching the brain was intact, and capillary depletion showed that most of it reached the parenchyma rather than being bound or loosely associated with the vasculature. Moreover, the low octanol/buffer partition coefficient indicated that lipophilicity was not an important factor in the blood-to-brain passage of mahogany (1377-1428).

Unlike mahogany (1377-1428), a saturable transport system from blood to brain does not exist for most of the recently described ingestive peptides. Although leptin, a larger polypeptide, enters the brain by a saturable system (Banks et al., 1996; Kastin et al., 1999b), this does not occur for orexin A or B (Kastin and Akerstrom, 1999c), cocaine- and amphetamine-related transcript (55-102) (Kastin and Akerstrom, 1999a), AgRP (83-132) (Kastin et al., 2000a), MCH (Kastin et al., 2000b), corticotropin-releasing hormone (Martins et al., 1996, 1997), or neuropeptide Y (Kastin and Akerstrom, 1999b). For each of these substances, the evidence for their influence on food ingestion was the impetus for investigation of their crossing of the BBB. By contrast, for mahogany (1377-1428), the order of investigation probably will be different, with demonstration of its crossing of the BBB serving as a stimulus for investigation of its effects on food ingestion. This is particularly relevant because of the finding described here of a transport system that is saturable and therefore susceptible to regulation.