Advertisement
Research ArticleNEUROPHARMACOLOGY

Extent and Direction of Ghrelin Transport Across the Blood-Brain Barrier Is Determined by Its Unique Primary Structure

William A. Banks, Matthias Tschöp, Sandra M. Robinson and Mark L. Heiman
Journal of Pharmacology and Experimental Therapeutics August 2002, 302 (2) 822-827; DOI: https://doi.org/10.1124/jpet.102.034827

Abstract

The novel hormone ghrelin is a potent orexigen that may counterbalance leptin. Ghrelin is the only secreted molecule requiring post-translational acylation with octanoic acid to ensure bioactivity. Ghrelin, predominantly derived from the stomach, may target neuroendocrine networks within the central nervous system (CNS) to regulate energy homeostasis. This would require ghrelin to cross the blood-brain barrier (BBB). In mice, we examined whether ghrelin crosses the BBB and whether its lipophilic side chain is involved in this process. We found that saturable systems transported human ghrelin from brain-to-blood and from blood-to-brain. Mouse ghrelin, differing from human ghrelin by two amino acids, was a substrate for the brain-to-blood but not for the blood-to-brain transporter and so entered the brain to a far lesser degree. des-Octanoyl ghrelin entered the brain by nonsaturable transmembrane diffusion and was sequestered once within the CNS. In summary, we show that ghrelin transport across the BBB is a complex, highly regulated bidirectional process. The direction and extent of passage are determined by the primary structure of ghrelin, defining a new role for the unique post-translational octanoylation.

Ghrelin, a novel 28 residue peptide hormone, has recently been identified as an endogenous ligand of the growth-hormone secretagogue receptor and is characterized by a novel post-translational acylation that ensures bioactivity (Kojima et al., 1999). Ghrelin is secreted in substantial amounts into circulation by the stomach and the intestine, whereas the CNS, as well as kidneys, placenta, pancreas, and pituitary, might produce miniscule amounts of ghrelin (Horvath et al., 2001).

Ghrelin acts peripherally and in the brain to regulate growth hormone secretion (Arvat et al., 2001), energy homeostasis (Tschop et al., 2000), and other physiological processes (Bowers, 2001). Crucial targets of the anabolic action of ghrelin are agouti-related protein/neuropeptide Y neurons located in the arcuate nucleus (Tschop et al., 2002). It is debatable if substances entering the nearby median eminence, a circumventricular organ (CVO) with a deficient blood-brain barrier (BBB), could leak into the arcuate nucleus (Horvath et al., 2001). Some evidence, however, shows that the outer layer of cells comprising the CVOs form a barrier preventing diffusion between CVOs and the rest of the brain (Maness et al., 1995).

Other neurons regulating energy balance that are targeted by ghrelin are located in CNS areas that are clearly protected by the BBB (Guan et al., 1997). This has raised the question of whether ghrelin is readily transported across BBB. It was once widely assumed that peptides and proteins could not cross the BBB. However, a number of peripheral hormones that participate in the regulation of energy have been shown to cross the BBB by saturable or nonsaturable mechanisms. Examples include leptin, insulin, and amylin (Baura et al., 1993; Banks et al., 1996; Banks and Kastin, 1998). Current efforts to better understand the physiological role of ghrelin have raised two major questions. Does peripheral ghrelin from the gastrointestinal tract act in the brain, and what is the functional role of the unique octanoyl side chain at Ser3? Therefore, we examined the ability of three forms of ghrelin to cross the BBB of the mouse: human ghrelin (h-ghrelin), mouse ghrelin (m-ghrelin), and des-octanoyl mouse ghrelin (des-m-ghrelin).

Materials and Methods

Source and Radiolabeling of Peptides.

m-Ghrelin, h-ghrelin, and des-m-ghrelin (Kojima et al., 1999) were purchased from Phoenix Pharmaceuticals, Inc. (Phoenix, TX). Both m-ghrelin and h-ghrelin were tested previously for acute orexigenic effects in mice in our laboratories to ensure bioactivity. Orexigenic potency of the used bioactive ghrelin molecules did not differ from the potency of ghrelin that has been synthesized in our own laboratories (Tschop et al., 2000). des-m-Ghrelin was not bioactive, as expected based on earlier publications (Kojima et al., 1999). Peptides were radioactively labeled with 131I by the chloramine-T method and purified by reverse-phase high-performance liquid chromatography on a C18 column.

Measurement of Octanol: Buffer Partition Coefficient.

Lipid solubilities of the radioactive-labeled ghrelin peptides were measured by determining their octanol/buffer partition coefficients. Radioactive ghrelin (1.5 × 105 cpm) was added to 1 ml of 0.25 M chloride-free phosphate buffer solution and 1 ml of octanol. This was vigorously mixed for 1 min, and the two phases were separated by centrifugation at 4500g for 10 min. Aliquots of 100 μl were taken in triplicate from each phase and counted. The mean partition coefficient was expressed as the log of the ratio of counts per minute (the octanol phase) to counts per minute (phosphate buffer solution phase).

Measurements of Ghrelin Uptake by Mouse Brain.

Multiple-time regression analysis (Blasberg et al., 1983; Banks and Kastin, 1990) was used to calculate the blood-to-brain unidirectional influx rate (Ki). The brain/serum ratios were plotted against their respective exposure times (Expt). Expt was calculated from the formula: Expt = [ 0t Cp(τ)dτ]/Cpt, where Cp is the level of radioactivity in serum and Cpt is the level of radioactivity in serum at time t. Expt corrects for the clearance of peptide from the blood.Ki, with its error term, is measured as the slope for the linear portion of the relation between the brain/serum ratios and Expt, and the y-intercept of the linearity measures Vi, the distribution volume, in brain at t = 0, so that the equation describing the linear portion of the relation between brain/serum ratios and Expt is: brain/serum ratio =Ki(Expt) +Vi.

The percentage of the intravenously injected dose taken up into each gram of mouse brain (%Inj/g) was calculated by multiplying the parenchymal values (in microliters per gram) by the percentage of the injected dose found in a microliter of blood. For competition studies, brain/serum ratios were calculated 10 min after the i.v. injection of131I-ghrelin with or without 1, 3, 10, or 30 μg/mouse unlabeled ghrelin.

Characterization of Radioactivity Extracted from Brain and Blood in Mice.

Radioactivity recovered from brain and serum was characterized by acid precipitation. The whole brains from mice that had received 131I-ghrelin were homogenized for each time point studied in 2 ml of 0.03 M bicarbonate buffer, containing 10 mM EDTA and 10 mM l-thyroxine, using a glass tissue grinder and 10 vertical strokes of the pestle. The homogenate was centrifuged at 14,000 rpm for 20 min, and the supernatant was collected. The pellet and supernatant were counted in a gamma counter (PerkinElmer Wallac, Inc., Gaithersburg, MD), and extraction was measured as the percentage of radioactivity in the supernatant. To determine degradation of 131I-ghrelin that occurred ex vivo (processing controls), 100 μl of131I-ghrelin in lactated Ringer's and BSA (LR-BSA) solution was placed on the surface of a nonradioactive mouse brain or in a tube used to obtain carotid blood, and the samples processed as above. For acid precipitation, 0.5 ml of brain homogenate was combined with 0.5 ml of 30% trichloroacetic acid, or 10 μl of serum was added to 0.50 ml of LR-BSA and 0.5 ml of 30% trichloroacetic acid. These mixtures were vigorously mixed, centrifuged at 5000g × 10 min, and the resulting supernatant and pellet collected. The percentage of radioactivity precipitated by acid was calculated as the percentage of total counts per minute (supernatant + pellet counts per minute) found in the pellet. Results for the brain and blood samples obtained after i.v. injection of131I-ghrelin were expressed as a percentage of the results for the processing controls. The raw counts per minute in brain and serum were multiplied by the percentage of acid precipitable radioactivity. All results reported here have been corrected for acid precipitation.

Capillary Depletion in Mice.

To determine whether the ghrelin peptides completely crossed the BBB, we performed capillary depletion as adapted to mice (Triguero et al., 1990; Gutierrez et al., 1993). CD-1 male mice anesthetized with i.p. urethane received an i.v. tracer injection of 0.2 ml of LR-BSA and 105 cpm of an I-ghrelin. At either 2 or 10 min after i.v. injection, blood from the abdominal aorta was collected. The thorax was opened, the descending aorta clamped, the jugular veins severed, and the vascular space of the brain washed free of blood by perfusing 20 ml of LR-BSA through the left ventricle of the heart. The cerebral cortex was removed, weighed, and emulsified with a glass homogenizer (10 strokes) in 0.8 ml of physiological buffer (10 mM HEPES, 141 mM NaCl, 4 mM KCl, 2.8 mM CaCl2, 1 mM MgSO4, 1 mM NaH2PO4, and 10 mMd-glucose adjusted to pH 7.4). Dextran solution (1.6 ml of a 26% solution) was added to the homogenate, which was thoroughly mixed, and homogenized again (3 strokes). Homogenization was performed at 4°C in less than 1 min. An aliquot of the homogenate was centrifuged at 5400g for 15 min at 4°C in a Beckman Allegra 21R centrifuge with a swinging bucket rotor (Beckman Coulter, Inc., Fullerton, CA). The pellet containing the brain vasculature and the supernatant containing the brain parenchyma were carefully separated, and the level of 131I determined in a gamma counter. The fractions were expressed as volumes of distribution in microliters per gram.

Mouse Brain Perfusion.

Mice were anesthetized with i.p. urethane, the thorax was opened, and the heart was exposed (Banks et al., 2000). Both jugular veins were severed, and the descending thoracic aorta was clamped. A 26-gauge butterfly needle was inserted into the left ventricle of the heart and Zlokovic's buffer (7.19 g/l NaCl, 0.3 g/l KCl, 0.28 g/l CaCl2, 2.1 g/l NaHCO3, 0.16 g/l KH2PO4, 0.17 g/l anhydrous MgCl2, 0.99 g/l d-glucose, and 10 g/l bovine serum albumin added the day of perfusion) containing m-ghrelin infused at a rate of 2 ml/min for 5 min. After perfusion, the needle was removed, and the mouse was decapitated. Brain/perfusion ratios were calculated by dividing the counts per minute/brain by the weight in grams of the brain and by the counts per minute in 1 μl of perfusion fluid to yield units of microliters per gram. The perfused brain remains viable for about 7 h (Krieglstein et al., 1972), and the BBB remains intact until about 12 h after death (Broman, 1950;Gröntoft, 1954).

Measurement of Brain-to-Blood efflux.

A method previously described that accurately quantifies efflux rates was used (Banks and Kastin, 1989). Mice were anesthetized with i.p. urethane, the scalp removed, and a hole made through the cranium 1.0 mm lateral and 1.0 mm posterior to the bregma with a 26-gauge needle. Tubing covered all but the terminal 2.5 to 3.0 mm of the needle so that the tip of the needle penetrated the brain tissue forming the roof of the lateral ventricle but did not penetrate its floor. One microliter of LR-BSA containing 5000 cpm of ghrelin was injected into the lateral ventricle with a 1-μl Hamilton syringe. To determine the rate of efflux, three mice were decapitated at each time of 2, 5, 10, and 20 min, and the residual radioactivity in the brain was determined. The amount of radioactivity in the brain at t = 0 was estimated as previously described in mice (n = 3) that had been overdosed with urethane. The log of the percentage of injected counts remaining in the whole brain was plotted against time and the slope used to calculate the half-time disappearance rate. To determine whether there was a saturable component to the retention of ghrelins by brain after i.c.v. injection, other mice received an i.c.v. injection containing a combination of radioactive and unlabeled ghrelins so that between 0.35 and 12.5 μg/mouse of ghrelin was injected. The mice were decapitated 10 min after injection, and the residual radioactivity was expressed as the percentage of the injection remaining in brain.

Statistics.

Means are reported with their standard errors and were compared by analysis of variance followed by a Newman-Keuls range test. Regression lines were computed by the least-squares method and compared statistically with the Prism 3.0 program (GraphPad, Inc., San Diego, CA).

Results

Acid precipitation of radioactivity recovered from brain and serum samples showed accumulation of radioactive degradation products with time. At the latest time point, as much as 60% of the radioactivity recovered from brain for all three ghrelin peptides was not precipitated by acid. Therefore, all brain and serum results presented here have been corrected to represent only radioactivity that was precipitated by acid. This tends to produce uptake rates that are conservative because radioactivity that degraded after entering the brain as intact peptide is excluded from calculations.

The blood to brain unidirectional influx rate (Ki) for131I-h-ghrelin was 0.324 ± 0.047 μl/g min and a Vi = 9.61 ± 0.46 μl/g (Fig. 1; n = 14,r = 0.926, p ≪ 0.01). Capillary depletion with washout of the vascular space showed that131I-h-ghrelin completely crossed the BBB, with only about 3 to 10% of the ghrelin taken up by brain being retained by capillaries at 2 and 10 min (Fig. 2).

Figure 1
Figure 1

Blood-to-brain influx of ghrelin peptides. Unidirectional influx rates were measurable for human and des-octanoyl mouse ghrelin but only to a minimal extent for mouse ghrelin. This and all other results have been corrected for acid precipitation.

Figure 2
Figure 2

Capillary depletion of ghrelin peptides. Complete penetration of the BBB by all three ghrelins was demonstrated by detection in the parenchymal (Parench) fractions. The small amounts of human ghrelin that were detected in the capillary fractions at 2 and 10 min for human ghrelin probably represent material that is being transported across the BBB.

The %Inj/g was 0.063 ± 0.003 for131I-h-ghrelin 10 min after injection (Table 1). Transport of radioactive h-ghrelin into brain was inhibited by coinjecting 10 and 30 μg/mouse of unlabeled h-ghrelin (analysis of variance: F(4,32) = 10.98; p ≪ 0.01; Fig. 3). Doses of 1 and 3 μg/mouse did not inhibit ghrelin uptake. In multiple studies, the Ki for m-ghrelin did not reach statistical significance (Fig. 1), indicating a very low penetration across the BBB. Capillary depletion showed no m-ghrelin retained by the capillaries, and the parenchyma contained less than 15% of the level measured for h-ghrelin (Fig. 2). The %Inj/g was 0.01 at 10 min (Table 1). The uptake by brain was not inhibited by coinjecting 30 μg/mouse of unlabeled m-ghrelin.

View this table:
Table 1

Comparison of m-ghrelin, des-m-ghrelin, and h-ghrelin

Figure 3
Figure 3

Passage of human ghrelin across the BBB by a saturable transporter after an i.v. injection. The brain/serum ratio measured for 131I-h-ghrelin 10 min after i.v. injection was decreased when 10 or 30 μg/mouse of unlabeled ghrelin was included in the i.v. injection. Similar experiments for m-ghrelin and des-octanoyl-m-ghrelin failed to detect a saturable component. ★,p < 0.01.

Brain perfusion of m-ghrelin for 5 min produced a brain/serum ratio 11.9 ± 2.1 (n = 5), a value that is largely accounted for by vascular space. This low level of uptake with perfusion shows that the poor uptake of m-ghrelin by brain cannot be explained by degradation in blood, saturation by endogenous circulating ghrelin, or binding to circulating proteins. des-m-Ghrelin had aKi = 0.573 ± 0.102 μl/g min and a Vi = 11.0 ± 1.5 μl/g (Fig. 1; n = 12, r = 0.876,p ≪ 0.01). Capillary depletion (Fig. 2) at 10 min showed a level in parenchyma intermediate between that of human and m-ghrelin.

The %Inj/g for des-m-ghrelin showed a similar pattern (Table 1). The presence of the octanoyl side chain made m-ghrelin about 5 times more lipid soluble (Table 1) but did not improve passage across the BBB, as speculated earlier (Horvath et al., 2001). The uptake by brain was not inhibited by coinjecting 30 μg/mouse of unlabeled des-m-ghrelin, indicating that passage across the BBB was probably based on the nonsaturable mechanism of transmembrane diffusion.

To further evaluate degradation, we analyzed radioactivity extracted from brain by high-performance liquid chromatography after administration of h-ghrelin by the intravenous and perfusion methods (Fig. 4). Intact ghrelin accounted for the majority of radioactivity in all brain samples, showing that it is intact ghrelin, which crosses the BBB. Efflux from the CNS was assessed by a quantitative method (Banks and Kastin, 1989) after i.c.v. injection of the radioactively labeled ghrelin molecules. The injected material distributes to both the CSF and paraventricular brain tissue, thus allowing a composite assessment of transport by both the choroid plexus and vascular barriers (Banks and Kastin, 1989). m-Ghrelin and h-ghrelin were both transported out of the CNS with half-time clearance rates of 16.4 and 12.3 min, respectively (Fig.5a). These rates exceed rates that could be explained by the reabsorption of CSF, which is usually about 25 to 45 min when measured with radioactive albumin. This efflux was based on a saturable process (Fig. 5c), with transport of m-ghrelin saturating at about a 2-fold lower dose than h-ghrelin (Fig. 5d). About 10 μg of either species of ghrelin produced maximal inhibition (Fig. 5d). des-m-Ghrelin was not cleared from the brain during the time of study by either saturable or nonsaturable processes (Fig. 5b). The lack of even the modest clearance attributable to CSF reabsorption suggests that des-m-ghrelin is retained by brain tissue in a manner similar to wheat germ agglutinin and insulin (Banks and Broadwell, 1994; Cashion et al., 1996).

Figure 4
Figure 4

Characterization of radioactivity extracted from brain after the i.v. injection of 131I-h-ghrelin. A, the processing control; B and C, results obtained 2 and 10 min after i.v. injection. D, data from a brain that has been perfused for 5 min and in which the vascular space has subsequently been washed free of blood.

Figure 5
Figure 5

Brain-to-blood efflux of ghrelin peptides. A shows that mouse and human ghrelin molecules were removed from the CNS at similar rates. B shows that des-octanoyl mouse ghrelin cannot cross the blood-brain barrier in the brain-to-blood direction. Since the relation between residual radioactivity and time was not statistically significant, the clearance rate cannot be measured. C shows an example of an inhibition curve for h-ghrelin in which efflux is inhibited when 10 times more ghrelin was administered. D compares inhibition curves for m-ghrelin and h-ghrelin. For all figures, n = 3/point.

Discussion

Leptin is a powerful modulator of body fat (Friedman and Halaas, 1998), with most obesity caused by a resistance to it (Heymsfield et al., 1999) probably mediated through impaired leptin transport across the blood-brain barrier (Banks et al., 1999) and impaired receptor function (Caro et al., 1996). Ghrelin, a more recently discovered peripheral regulator of energy homeostasis, seems to complement leptin in signaling the CNS about the current state of energy balance (Horvath et al., 2001). Since circulating ghrelin is derived from the stomach and its centrally located target neurons seem to be similar, if not identical, to those targeted by leptin, the BBB may play an important role for ghrelin action as well.

To investigate the central action of the peripheral hormone ghrelin, we studied the ability of three ghrelin peptides (human, mouse, and mouse des-octanoyl) to cross the BBB of the mouse in the brain-to-blood and blood-to-brain directions. We found that each of these molecules crossed the BBB but differed in degree of passage, direction of passage, and transport mechanism. h-Ghrelin was readily transported by a saturable system across the BBB in both directions, whereas the transport of m-ghrelin was saturable in only the brain-to-blood direction. m-Ghrelin differs from h-ghrelin in two of its 28 residues, with lysine replacing arginine at position 11 and alanine replacing valine at position 122. These two amino acids are, therefore, critical for recognition by the blood-to-brain transporter but not the CNS-to-blood transporter. The low uptake of m-ghrelin by brain after i.v. injection is probably caused by the combination of saturable efflux without saturable influx.

In contrast, the accumulation of des-m-ghrelin resulted from lack of efflux and by retention by brain tissue once within the CNS. Retention by brain after i.c.v. injection has been seen previously for other substances (Banks and Broadwell, 1994; Cashion et al., 1996) and usually indicates that those molecules are sequestered and internalized by the periventricular tissues (Banks and Broadwell, 1994). Saturable and nonsaturable passage and brain-to-blood and blood-to-brain passage of peptides and regulatory proteins have been shown to be regulated by physiological and pathological processes. As such, uptake rates can vary. The influx of h-ghrelin always exceeded that of m-ghrelin, but des-ghrelin was more variable. In addition, m-ghrelin could be occasionally demonstrated by capillary depletion and by multiple-time regression analysis to cross the BBB, although uptake was not clearly detectable. These observations raise the possibility that the BBB mechanisms regulating ghrelin accumulation by brain may be influenced by pathophysiological events, just as is the case for insulin and leptin (Florant et al., 1991; Banks et al., 1999).

In conclusion, these results demonstrate a saturable transport system directed in the CNS-to-blood direction, which has a similar affinity for m-ghrelin and h-ghrelin and requires the presence of the unique octanoyl component of the ghrelin molecule. Furthermore, we showed the existence of a saturable transporter for the blood-to-brain direction, which recognizes h-ghrelin but not m-ghrelin. des-m-Ghrelin also crosses the BBB in the blood to brain direction but by a nonsaturable mechanism (Fig. 6). These complex mechanisms are likely to be a regulatory component balancing central ghrelin action. The removal of ghrelin from the brain rather than its transport into the CNS adds new impact to the question, if and in which quantities ghrelin is produced in the brain. If the hypothalamus is a relevant source of ghrelin, then the clearance of bioactive ghrelin from the brain across the BBB could be an important regulatory mechanism to balance energy homeostasis and the secretion of growth hormone. Similar to leptin, ghrelin levels are found to be increased in disease states where a direct etiological role would have suggested the opposite (Considine et al., 1996; Friedman and Halaas, 1998; Horvath et al., 2001; Otto et al., 2001). Patients with anorexia nervosa, for example, exhibit significantly increased plasma levels of ghrelin (Otto et al., 2001). Further studies are necessary to investigate the existence of ghrelin resistance and whether BBB transport mechanisms might be causally involved. A more widespread occurrence of the herein described differential transport mechanism across the BBB and the possible existence of other signaling molecules carrying octanoyl side chains (Kojima et al., 1999; Bowers, 2001) remain to be shown.

Figure 6
Figure 6

Differential transport of mouse ghrelin, des-octanoyl mouse ghrelin, and human ghrelin across the blood-brain barrier in mice. Although octanoylated (bioactive) mouse ghrelin crosses the mouse BBB predominantly in the brain-to-blood direction, passage for des-octanoyl mouse ghrelin was observed only in the blood-to-brain direction. Human ghrelin, which differs from mouse ghrelin by two amino residues only, was transported in both directions in mice. The extent and direction in which the ghrelin can cross the BBB is therefore influenced by at least two features of its primary structure, its post-translationally added fatty acid side chain and its amino acid sequence.

Footnotes

  • Supported by Veterans Affairs, R01 NS41863, R01AA12743, and Eli Lilly and Company.

  • DOI: 10.1124/jpet.102.034827

  • Abbreviations:
    CNS
    central nervous system
    CVO
    circumventricular organ
    BBB
    the blood-brain barrier
    h-ghrelin
    human ghrelin
    m-ghrelin
    mouse ghrelin
    des-m-ghrelin
    des-octanoyl mouse ghrelin
    %Inj/g
    percentage of the intravenously injected dose taken up into each gram of mouse brain
    LR-BSA
    lactated Ringer's and bovine serum albumin solution
    CSF
    cerebrospinal fluid
    • Received February 19, 2002.
    • Accepted March 27, 2002.

References

  1. ↵
    1. Arvat E,
    2. Maccario M,
    3. Di Vito L,
    4. Broglio F,
    5. Benso A,
    6. Gottero C,
    7. Papotti M,
    8. Muccioli G,
    9. Dieguez C,
    10. Casamieva FF,
    11. et al.
    (2001) Endocrine activities of ghrelin a natural growth hormone secretagogue (GHS) in humans: comparison and interactions with hexarelin a nonnatural peptidyl GHS and GH-releasing hormone. J Clin Endocrinol Metab 86:1169–1174.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Banks WA,
    2. Broadwell RD
    (1994) Blood to brain and brain to blood passage of native horseradish peroxidase, wheat germ agglutinin and albumin: pharmacokinetic and morphological assessments. J Neurochem 62:2404–2419.
    OpenUrlPubMed
  3. ↵
    1. Banks WA,
    2. Clever CM,
    3. Farrell CL
    (2000) Partial saturation and regional variation in the blood to brain transport of leptin in normal weight mice. Am J Physiol 278:E1158–E1165.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    1. Banks WA,
    2. DiPalma CR,
    3. Farrell CL
    (1999) Impaired transport of leptin across the blood-brain barrier in obesity. Peptides 20:1341–1345.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Conn PM
    1. Banks WA,
    2. Kastin AJ
    (1989) Quantifying carrier-mediated transport of peptides from the brain to the blood. in Methods Enzymol, ed Conn PM (Academic Press, San Diego), 168:652–660.
  6. ↵
    1. Porter JC,
    2. Jezova D
    1. Banks WA,
    2. Kastin AJ
    (1990) Exchange of peptides between the circulation and the nervous system: role of the blood-brain barrier. in Circulating Regulatory Factors and Neuroendocrine Function, Advances in Experimental Biological Medicine, eds Porter JC, Jezova D (Plenum Press, New York), 274:59–69.
  7. ↵
    1. Banks WA,
    2. Kastin AJ
    (1998) Differential permeability of the blood-brain barrier to two pancreatic peptides: insulin and amylin. Peptides 19:883–889.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Banks WA,
    2. Kastin AJ,
    3. Huang W,
    4. Jaspan JB,
    5. Maness LM
    (1996) Leptin enters the brain by a saturable system independent of insulin. Peptides 17:305–311.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Baura GD,
    2. Foster DM,
    3. Porte D, Jr,
    4. Kahn SE,
    5. Bergman RN,
    6. Cobelli C,
    7. Schwartz MW
    (1993) Saturable transport of insulin from plasma into the central nervous system of dogs in vivo: a mechanism for regulated insulin delivery to the brain. J Clin Invest 92:1824–1830.
  10. ↵
    1. Blasberg RG,
    2. Fenstermacher JD,
    3. Patlak CS
    (1983) Transport of α-aminoisobutyric acid across brain capillary and cellular membranes. J Cereb Blood Flow Metab 3:8–32.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Bowers CL
    (2001) Unnatural growth hormone-releasing peptide begets natural ghrelin. J Clin Endocrin Metab 86:1464–1469.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Broman T
    (1950) Supravital analysis of disorders in the cerebrovascular permeability. Arch Psychiatr 25:19–31.
    OpenUrl
  13. ↵
    1. Caro JF,
    2. Kolaczynski JW,
    3. Nyce MR,
    4. Ohannesian JP,
    5. Opentanova I,
    6. Goldman WH,
    7. Lynn RB,
    8. Zhang P-L,
    9. Sinha MD,
    10. Considine RV
    (1996) Decreased cerebrospinal-fluid/serum leptin ratio in obesity: a possible mechanism for leptin resistance. Lancet 348:159–161.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Cashion MF,
    2. Banks WA,
    3. Kastin AJ
    (1996) Sequestration of centrally administered insulin by the brain: effects of starvation, aluminum and TNF-α. Hormone Behav 30:280–286.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Considine RV,
    2. Sinha MK,
    3. Heiman ML,
    4. Kriauciunas A,
    5. Stephens TW,
    6. Nyce MR,
    7. Ohannesian JP,
    8. Marco CC,
    9. McKee LJ,
    10. Bauer TL,
    11. Caro JF
    (1996) Serum immunoreactive-leptin concentrations in normal-weight and obese humans. N Engl J Med 334:292–295.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Florant GL,
    2. Richardson RD,
    3. Mahan S,
    4. Singer L,
    5. Woods SC
    (1991) Seasonal changes in CSF insulin levels in marmots: insulin may not be a satiety signal for fasting in winter. Am J Physiol 260:R712–R716.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Friedman JM,
    2. Halaas JL
    (1998) Leptin and the regulation of body weight in mammals. Nature (Lond) 395:763–770.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Gröntoft O
    (1954) Intracranial haemorrhage and blood-brain barrier problems in the new-born. Acta Pathol Microbiol Scand 54 (Suppl C) 1–109.
    OpenUrl
  19. ↵
    1. Guan XM,
    2. Yu H,
    3. Palyha OC,
    4. McKee KK,
    5. Feighner SD,
    6. Sirinathsinghji DJ,
    7. Smith RG,
    8. Van der Ploeg LH,
    9. Howard AD
    (1997) Distribution of mRNA encoding the growth hormone secretagogue receptor in brain and peripheral tissues. Brain Res Mol Brain Res 48:23–29.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Gutierrez EG,
    2. Banks WA,
    3. Kastin AJ
    (1993) Murine tumor necrosis factor-α is transported from blood to brain in the mouse. J Neuroimmunol 47:169–176.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Heymsfield SB,
    2. Greenberg AS,
    3. Fujioka K,
    4. Dixon RM,
    5. Kushner R,
    6. Hunt T,
    7. Lubina JA,
    8. Patane J,
    9. Self B,
    10. Hunt P,
    11. McCamish M
    (1999) Recombinant leptin for weight loss in obese and lean adults: a randomized, controlled, dose-escalation trial. J Am Med Assoc 282:1568–1575.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Horvath TL,
    2. Diano S,
    3. Sotonyi P,
    4. Heiman M,
    5. Tschop M
    (2001) Minireview: ghrelin and the regulation of energy balance—a hypothalamic perspective. Endocrinology 142:4163–4169.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Kojima M,
    2. Hosoda H,
    3. Date Y,
    4. Nakazato M,
    5. Matsuo H,
    6. Kangawa K
    (1999) Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature (Lond) 402:656–660.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Krieglstein G,
    2. Krieglstein J,
    3. Urban W
    (1972) Long survival time of an isolated perfused rat brain. J Neurochem 19:885–886.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Maness LM,
    2. Banks WA,
    3. Zadina JE,
    4. Kastin AJ
    (1995) Selective transport of blood-borne interleukin-1α into the posterior division of the septum of the mouse brain. Brain Res 700:83–88.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Otto B,
    2. Cuntz U,
    3. Fruehauf E,
    4. Wawarta R,
    5. Folwaczny C,
    6. Riepl RL,
    7. Heiman ML,
    8. Lehnert P,
    9. Fichter M,
    10. Tschop M
    (2001) Weight gain decreases elevated plasma ghrelin concentrations of patients with anorexia nervosa. Eu J Endocrin 145:669–673.
  27. ↵
    1. Triguero D,
    2. Buciak J,
    3. Pardridge WM
    (1990) Capillary depletion method for quantification of blood-brain barrier transport of circulating peptides and plasma proteins. J Neurochem 54:1882–1888.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Tschop M,
    2. Smiley DL,
    3. Heiman ML
    (2000) Ghrelin induces adiposity in rodents. Nature (Lond) 407:908–913.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Tschop M,
    2. Statnick MA,
    3. Suter TM,
    4. Heiman ML
    (2002) GH-releasing peptide-2 increases fat mass in mice lacking NPY: indication for a crucial mediating role of hypothalamic agouti-related protein. Endocrinology 143:558–68.
    OpenUrlCrossRefPubMed
Back to top

In this issue

Download PDF
Article Alerts
Email Article
Citation Tools

Research ArticleNEUROPHARMACOLOGY

Extent and Direction of Ghrelin Transport Across the Blood-Brain Barrier Is Determined by Its Unique Primary Structure

William A. Banks, Matthias Tschöp, Sandra M. Robinson and Mark L. Heiman
Journal of Pharmacology and Experimental Therapeutics August 1, 2002, 302 (2) 822-827; DOI: https://doi.org/10.1124/jpet.102.034827
Twitter logo Facebook logo Mendeley logo

Jump to section

Advertisement