Impaired transport of leptin across the blood-brain barrier in obesity

Leptin is a 17-kDa protein secreted by fat cells that regulates body adiposity by crossing the blood-brain barrier (BBB) to affect feeding and thermogenesis. Obese human and rodent models of dietary obesity have shown decreased sensitivity to blood-borne leptin, postulated to be due to impaired transport of leptin across the BBB. We show here that the transport rate of leptin across the BBB is reduced about 2/3 in 12-month-old obese CD-1 mice. In a follow-up study, a perfusion method was used that replaced the blood with a buffer containing low concentrations of radioactive leptin. Obese mice still had lower rates of transport into the brain than lean mice, which shows that the reduction in transport rate associated with obesity is not due simply to saturation of transporter secondary to higher serum leptin levels as has been thought, but to a decreased capacity of the BBB to transport leptin. This suggests a new model for obesity in which a defect in the BBB transport of leptin into the CNS underlies the insensitivity to leptin and leads to obesity.     

Blood-brain barrier is involved in the efflux transport of a neuroactive steroid, dehydroepiandrosterone sulfate, via organic anion transporting polypeptide 2

We have investigated the transport characteristics of dehydroepiandrosterone sulfate (DHEAS), a neuroactive steroid, at the blood-brain barrier (BBB) in a series of functional in vivo and in vitro studies. The apparent BBB efflux rate constant of [(3)H]DHEAS evaluated by the brain efflux index method was 2.68 x 10(-2) min(-1). DHEAS efflux transport was a saturable process with a Michaelis constant (K:(m)) of 32.6 microM: Significant amounts of [(3)H]DHEAS were determined in the jugular venous plasma by HPLC, providing direct evidence that most of the DHEAS is transported in intact form from brain to the circulating blood across the BBB. This efflux transport of [(3)H]DHEAS was significantly inhibited by common rat organic anion-transporting polypeptide (oatp) substrates such as taurocholate, cholate, sulfobromophthalein, and estrone-3-sulfate. Moreover, the apparent efflux clearance of [(3)H]DHEAS across the BBB (118 microl/min-g of brain) was 10.4-fold greater than its influx clearance estimated by the in situ brain perfusion technique (11.4 microl/min-g of brain), suggesting that DHEAS is predominantly transported from the brain to blood across the BBB. In cellular uptake studies using a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4), [(3)H]DHEAS uptake by TM-BBB4 cells exhibited a concentration dependence with a K:(m) of 34.4 microM: and was significantly inhibited by the oatp2-specific substrate digoxin. Conversely, [(3)H]digoxin uptake by TM-BBB4 cells was significantly inhibited by DHEAS. Moreover, the net uptake of [(3)H]DHEAS at 30 min was significantly increased under ATP-depleted conditions, suggesting that an energy-dependent efflux process may also be involved in TM-BBB4. RT-PCR and sequence analysis suggest that an oatp2 is expressed in TM-BBB4 cells. In conclusion, DHEAS efflux transport takes place across the BBB, and studies involving in vitro DHEAS uptake and RT-PCR suggest that there is oatp2-mediated DHEAS transport at the BBB.     

Effect of middle cerebral artery occlusion on the passage of pituitary adenylate cyclase activating polypeptide across the blood-brain barrier in the rat

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to be a potent neuroprotective agent in global and focal ischemia. We demonstrated that PACAP could cross the blood-brain barrier (BBB) by a saturable transport system, and a systemic administration of PACAP reduced the infarct induced by unilateral middle cerebral artery occlusion (MCAO). Therefore, we studied whether this transport system is affected by MCAO in the rat. The entry of PACAP38 into the brain was compared in five groups: control, 4, 6, 24, and 48 h after MCAO. [(125)I]PACAP38 was injected intravenously and serum and various brain regions were collected 3 min later. The rate of entry into the brain of PACAP38 was also determined. We showed that PACAP entered the rat brain via a rapid transport system when the BBB is intact. After transient (2 h) unilateral MCAO, all regions of the brain, showed a selective increase in the passage of PACAP38 across the BBB after 4 h after the occlusion, which was not related to any generalized change in the permeability of the BBB, as measured with albumin. A significant decrease in the amount of PACAP38 entering the brain was observed in the 6- and 24-h groups, but it returned to the baseline level in the 48-h group. These results suggest that focal cerebral ischemia can selectively modify the passage of PACAP38 across the BBB, in both damaged and undamaged sides of the brain, and that these changes in influx are not solely due to the disruption of BBB. These findings imply the necessity of adjusting the dose of intravenously administered PACAP38 in order to maximize its therapeutic effect on the brain damage resulting from focal ischemia     

Niacinamide transport through the blood-brain barrier

The unidirectional influx of niacinamide across cerebral capillaries, the anatomical locus of the blood-brain barrier, was measured with an in situ rat brain perfusion technique employing [¹⁴C]niacinamide. Niacinamide was transported rapidly across the blood-brain barrier by a system that was not saturable with 10 mM niacinamide in the perfusate. However, with periods of perfusion longer than 30 seconds, there was substantial backflow of [¹⁴C]niacinamide into the perfusate. Niacinamide (1.7 M) transport through the blood-brain barrier was not significantly inhibited by 3-acetylpyridine. Thus, niacinamide is transported rapidly and bidirectionally through the blood-brain barrier by a high capacity transport system. Although involved in the transfer of niacinamide between blood and brain, this transport system does not play an important regulatory role in the synthesis of NMN, NAD, and NADP from niacinamide in brain.     

Blood-brain barrier transport of pramipexole, a dopamine D2 agonist

The blood-brain barrier (BBB) transport of pramipexole, a potent dopamine receptor agonist with high efficacy for Parkinson's disease, was mainly characterized using immortalized rat brain capillary endothelial cells (RBEC)1 as an in vitro BBB model. [(14)C]Pramipexole uptake by RBEC1 was dependent on temperature and pH, but not sodium ion concentration or membrane potential. The uptake was inhibited by several organic cations including pyrilamine. Mutual inhibition was observed between pramipexole and pyrilamine. In addition, [(14)C]pramipexole uptake was stimulated by preloading unlabeled pramipexole. RT-PCR analysis for organic cation transporters (rOCT1-3, rOCTN1-2) in RBEC1 was performed. The mRNA level of rOCTN2 was the highest, followed by rOCTN1, while expression of rOCT1, rOCT2 and rOCT3 was negligible. The brain uptake of [(14)C]pramipexole, which was measured by the in situ rat brain perfusion technique, was significantly inhibited by unlabeled pramipexole. These results suggest that pramipexole is, at least in part, transported across the BBB by an organic cation-sensitive transporter. The pramipexole transport in RBEC1 was pH-dependent, but sodium- and membrane potential-independent.     

Pantothenic Acid Transport Through the Blood-Brain Barrier

The unidirectional influx of D-pantothenic acid (PA) across cerebral capillaries, the anatomical locus of the blood-brain barrier, was measured with an in situ rat brain perfusion technique using [3H]D-PA (1.1 Ci/mmol). PA was transported across the blood-brain barrier by a saturable system that could be described by a Michaelis-Menten transport model with a half-saturation concentration and maximal influx rate of 19 microM and 0.21 nmol/g of brain/min, respectively. PA (0.3 microM) transport through the blood-brain barrier was significantly inhibited by probenecid, nonanoic acid, and biotin (all less than or equal to 0.25 mM), but not by penicillin G, pyruvate, beta-hydroxybutyrate, L-leucine (all 1 mM), or poly-L-lysine HBr (1 mg/ml). Probenecid (0.25 mM), nonanoic acid (0.5 mM), and PA (1.0 mM) did not inhibit [3H]L-leucine transport through the blood-brain barrier, whereas 30 microM-L-leucine inhibited [3H]leucine transport to 23% of control values. Thus, PA is transported through the blood-brain barrier by a low-capacity, saturable transport system with a half-saturation concentration approximately 10 times the plasma PA concentration. Although involved in the transfer of PA from blood into brain, this system does not play an important regulatory role in the synthesis of CoA from PA in brain.     

Influence of breast cancer resistance protein (Abcg2) and p-glycoprotein (Abcb1a) on the transport of imatinib mesylate (Gleevec) across the mouse blood-brain barrier

Imatinib, a protein tyrosine kinase inhibitor, may prevent the growth of glioblastoma cells. Unfortunately, its brain distribution is restricted by p-glycoprotein (p-gp or multidrug resistance protein Mdr1a), and probably by breast cancer resistance protein (Bcrp1), two efflux pumps expressed at the blood-brain barrier (BBB). We have used in situ brain perfusion to investigate the mechanisms of imatinib transport across the mouse BBB. The brain uptake of imatinib in wild-type mice was limited by saturable efflux processes. The inhibition of p-gp, by valspodar and zosuquidar, increased imatinib uptake (2.5-fold), as did the deficiency of p-gp in Mdr1a/1b(-/-) mice (5.5-fold). Perfusing imatinib with the p-gp/Bcrp1 inhibitor, elacridar, enhanced the brain uptake of imatinib in wild-type (4.1-fold) and Mdr1a/1b(-/-) mice (1.2-fold). However, the brain uptake of imatinib was similar in wild-type and Bcrp1(-/-) mice when it was perfused at a non-saturating concentration. The brain uptake of CGP74588, an active metabolite of imatinib, was low. It was increased by perfusion with elacridar (twofold), but not with valspodar and zosuquidar. CGP74588 uptake was 1.5 times greater in Bcrp1(-/-) mice than in wild-type mice. These data suggest that imatinib transport at the mouse BBB is limited by p-gp and probably by Bcrp1, and that CGP74588 transport is restricted by Bcrp1.     

Human immunodeficiency virus-1 uses the mannose-6-phosphate receptor to cross the blood-brain barrier

HIV-1 circulates both as free virus and within immune cells, with the level of free virus being predictive of clinical course. Both forms of HIV-1 cross the blood-brain barrier (BBB) and much progress has been made in understanding the mechanisms by which infected immune cells cross the blood-brain barrier BBB. How HIV-1 as free virus crosses the BBB is less clear as brain endothelial cells are CD4 and galactosylceramide negative. Here, we found that HIV-1 can use the mannose-6 phosphate receptor (M6PR) to cross the BBB. Brain perfusion studies showed that HIV-1 crossed the BBB of all brain regions consistent with the uniform distribution of M6PR. Ultrastructural studies showed HIV-1 crossed by a transcytotic pathway consistent with transport by M6PR. An in vitro model of the BBB was used to show that transport of HIV-1 was inhibited by mannose, mannan, and mannose-6 phosphate and that enzymatic removal of high mannose oligosaccharide residues from HIV-1 reduced transport. Wheatgerm agglutinin and protamine sulfate, substances known to greatly increase transcytosis of HIV-1 across the BBB in vivo, were shown to be active in the in vitro model and to act through a mannose-dependent mechanism. Transport was also cAMP and calcium-dependent, the latter suggesting that the cation-dependent member of the M6PR family mediates HIV-1 transport across the BBB. We conclude that M6PR is an important receptor used by HIV-1 to cross the BBB.     

Differential BBB interactions of three ingestive peptides: obestatin, ghrelin, and adiponectin

Endogenous compounds, including ingestive peptides, can interact with the blood-brain barrier (BBB) in different ways. Here we used in vivo and in vitro techniques to examine the BBB permeation of the newly described satiety peptide obestatin. The fate of obestatin in blood and at the BBB was contrasted with that of adiponectin. By the sensitive multiple time-regression method, obestatin appeared to have an extremely fast influx rate to the brain whereas adiponectin did not cross the BBB. HPLC analysis, however, showed the obestatin result to be spurious, reflecting rapid degradation. Absence of BBB permeation by obestatin and adiponectin was in contrast to the saturable transport of human ghrelin reported previously. As a positive control, ghrelin showed saturable binding and endocytosis in RBE4 cerebral microvessel endothelial cells. By comparison, obestatin lacked specific binding and endocytosis, and the small amount internalized showed rapid intracellular degradation before the radioactivity was released by exocytosis. The differential interactions of obestatin, adiponectin, and ghrelin with the BBB illustrate their distinctive physiological interactions with the CNS.     

Effects of cell-type specific leptin receptor mutation on leptin transport across the BBB

The functions of leptin receptors (LRs) are cell-type specific. At the blood-brain barrier, LRs mediate leptin transport that is essential for its CNS actions, and both endothelial and astrocytic LRs may be involved. To test this, we generated endothelia specific LR knockout (ELKO) and astrocyte specific LR knockout (ALKO) mice. ELKO mice were derived from a cross of Tie2-cre recombinase mice with LR-floxed mice, whereas ALKO mice were generated by a cross of GFAP-cre with LR-floxed mice, yielding mutant transmembrane LRs without signaling functions in endothelial cells and astrocytes, respectively. The ELKO mutation did not affect leptin half-life in blood or apparent influx rate to the brain and spinal cord, though there was an increase of brain parenchymal uptake of leptin after in situ brain perfusion. Similarly, the ALKO mutation did not affect blood-brain barrier permeation of leptin or its degradation in blood and brain. The results support our observation from cellular studies that membrane-bound truncated LRs are fully efficient in transporting leptin, and that basal levels of astrocytic LRs do not affect leptin transport across the endothelial monolayer. Nonetheless, the absence of leptin signaling at the BBB appears to enhance the availability of leptin to CNS parenchyma. The ELKO and ALKO mice provide new models to determine the dynamic regulation of leptin transport in metabolic and inflammatory disorders where cellular distribution of LRs is shifted.     

Role of the blood-brain barrier in the evolution of feeding and cognition

The blood-brain barrier (BBB) regulates the blood-to-brain passage of gastrointestinal hormones, thus informing the brain about feeding and nutritional status. Disruption of this communication results in dysregulation of feeding and body weight control. Leptin, which crosses the BBB to inform the CNS about adiposity, provides an example. Impaired leptin transport, especially coupled with central resistance, results in obesity. Various substances/conditions regulate leptin BBB transport. For example, triglycerides inhibit leptin transport. This may represent an evolutionary adaptation in that hypertriglyceridemia occurs during starvation. Inhibition of leptin, an anorectic, during starvation could have survival advantages. The large number of other substances that influence feeding is explained by the complexity of feeding. This complexity includes cognitive aspects; animals in the wild are faced with cost/benefit analyses to feed in the safest, most economical way. This cognitive aspect partially explains why so many feeding substances affect neurogenesis, neuroprotection, and cognition. The relation between triglycerides and cognition may be partially mediated through triglyceride's ability to regulate the BBB transport of cognitively active gastrointestinal hormones such as leptin, insulin, and ghrelin.     

Biotin Transport Through the Blood-Brain Barrier

The unidirectional influx of biotin across cerebral capillaries, the anatomical locus of the blood-brain barrier, was measured with an in situ rat brain perfusion technique employing [3H]biotin. Biotin was transported across the blood-brain barrier by a saturable system with a one-half saturation concentration of approximately 100 microM. The permeability-surface area products were 10(-4) s-1 with a biotin concentration of 0.02 microM in the perfusate. Probenecid, pantothenic acid, and nonanoic acid but not biocytin or biotin methylester (all 250 microM) inhibited biotin transfer through the blood-brain barrier. The isolated rabbit choroid plexus was unable to concentrate [3H]biotin from medium containing 1 nM [3H]biotin. These observations provide evidence that: biotin is transported through the blood-brain barrier by a saturable transport system that depends on a free carboxylic acid group, and the choroid plexus is probably not involved in the transfer of biotin between blood and cerebrospinal fluid.     

Saturable transport of the neurokinin-1 non-peptide antagonist LY303870 across the rat blood-brain barrier after intravenous administration

LY303870 (LY) is a non-peptide neurokinin-1 receptor antagonist that has effects on the brain after peripheral administration. We determined whether LY given by intravenous (iv) injection can cross the blood-brain barrier (BBB). Multiple-time regression analysis showed the unidirectional influx rate (Ki) from blood to brain for LY labeled with tritium to be 6.41+/-0.85 microl/g-min and influx was inhibited by unlabeled LY. HPLC and mass spectrometry showed LY was stable in blood and brain. LY reached a brain/serum ratio of 190+/-12 microl/g with about 0.07% of the injected dose entering each gram of brain. These results show that LY is transported across the BBB from serum into brain by a saturable system.     

Changes in dipole membrane potential at the mouse blood–brain barrier enhance the transport of 99mTechnetium Sestamibi more than inhibiting Abcb1, Abcc1, or Abcg2

Cationic ^99mTc-agents like ^99mTc-hexakis-2-methoxyisobutyl isonitrile (^99mTc-MIBI) cannot be used for brain imaging because they do not enter the brain as readily as some uncharged ^99mTc-compounds. The mechanism by which cationic ^99mTc-agents are transported across the blood–brain barrier (BBB) remains unclear. We explored ^99mTc-MIBI transport by in situ mouse brain perfusion to determine the influence of BBB features like the ATP-binding cassette transporters (Abcb1/P-glycoprotein (P-gp), Abcc1/Mrp1, and Abcg2/Bcrp), organic cation transporters (Slc22a1-3/Oct1-3), the transmembrane potential and the dipole membrane potential. P-gp reduced ^99mTc-MIBI transport across the BBB of P-gp-deficient mice 2.2-fold, as confirmed by PSC833 and GF120918 inhibition. Paradoxically verapamil decreased its transport '0.6-fold'. Reducing the BBB dipole membrane potential with tetraphenylborate or phloretin increased ^99mTc-MIBI transport about 12- and 20-fold, respectively. Guanidine, diphenhydramine, and carnitine significantly decreased ^99mTc-MIBI transport, but tetraethylammonium did not. ^99mTc-MIBI transport at the BBB is restricted by P-gp but not by Mrp1 or Bcrp. Some organic cations reduced the influx of ^99mTc-MIBI into the brain independently of Oct1, 2 and 3, but this could be due to their effect on another cation transporter. The membrane dipole potential of the luminal BBB membrane appeared to be the main factor restricting ^99mTc-MIBI permeability.     

Blood-brain barrier transport of a novel micro 1-specific opioid peptide,H-Tyr-D-Arg-Phe-beta-Ala-OH (TAPA)

The purpose of this study was to clarify the mechanism of the blood-brain barrier (BBB) transport of H-Tyr-D-Arg-Phe-beta-Ala-OH (TAPA), which is a novel dermorphin analog with high affinity for the micro 1-opioid receptor. The in vivo BBB permeation influx rate of [125I]TAPA after an i.v. bolus injection (7.3 pmol/g body weight) into mice was estimated to be 0.265 +/- 0.025 microL/(min.g of brain). The influx rate of [125I]TAPA was reduced 70% by the coadministration of unlabeled TAPA (33 nmol/g of brain), suggesting the existence of a specific transport system for TAPA at the BBB. In order to elucidate the BBB transport mechanism of TAPA, a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4) was used as an in vitro model of the BBB. The acid-resistant binding of [125I]TAPA, which represents the internalization of the peptide into cells, was temperature- and concentration-dependent with a half-saturation constant of 10.0 +/- 1.7 microm. The acid-resistant binding of TAPA was significantly inhibited by 2,4-dinitrophenol, dansylcadaverine (an endocytosis inhibitor) and poly-l-lysine and protamine (polycations). These results suggest that TAPA is transported through the BBB by adsorptive-mediated endocytosis, which is triggered by binding of the peptide to negatively charged sites on the surface of brain capillary endothelial cells. Blood-brain barrier transport via adsorptive-mediated endocytosis plays a key role in the expression of the potent opioid activity of TAPA in the CNS.     

Hypoxanthine transport through the blood-brain barrier

The unidirectional influx of hypoxanthine across cerebral capillaries, the anatomical locus of the blood=brain barrier, was measured with an in situ rat brain perfusion technique employing [³H]hypoxanthine. Hypoxanthine was transported across the blood-brain barrier by a saturable system with a one-half saturation concentration of approximately 0.4 mM. The permeability-surface area product was 3×10^–4 sec^–1 with a hypoxanthine concentration of 0.02 M in the perfusate. Adenine (4 mM) and uracil and theophylline (both 10 mM), but not inosine (10 mM) or leucine (1 mM), inhibited hypoxanthine transfer through the blood-brain barrier. Thus, hypoxanthine is transported through the blood-brain barrier by a high-capacity, saturable transport system with a half-saturation concentration about 100 times the plasma hypoxanthine concentration. Although involved in the transport hypoxanthine from blood into brain, this system is not powerful enough to transfer important quantities of hypoxanthine from blood into brain.     

Uptake and degradation of blood-borne insulin by the olfactory bulb.

Insulin found within the brain is derived from the blood and can affect various central nervous system (CNS) functions. The olfactory bulb contains one of the highest concentrations of insulin and insulin receptors within the CNS. To determine the mechanism underlying this high concentration of insulin, we used radioactively iodinated insulin to compare the blood to tissue transport rates and tissue degradation rates for the olfactory bulb, whole brain and spinal cord. We found that the olfactory bulb had both the highest transport rate across the blood-brain barrier (BBB) and the highest rate of degradation. Because a higher degradation rate would decrease, not increase, tissue concentrations of insulin, BBB transport may be the primary mechanism by which high concentrations of insulin are maintained within the olfactory bulb. This illustrates an adaptive aspect of the BBB in its regulation of the exchange of information molecules between the blood and the CNS.     

Effect of cardiac arrest on brain weight and the permeability of the blood-brain and blood-spinal cord barrier to albumin and tumor necrosis factor-alpha

Time-dependent changes in brain and spinal cord were studied in mice in a cardiac arrest model. A transient decrease in body weight and a prolonged decrease in brain weight occurred after arrest whereas spinal cord weight was unchanged. The permeability of the blood-brain barrier (BBB) to I131-albumin and I131 tumor necrosis factor-alpha (TNF) showed maximal, non-significant increases on day 5 after cardiac arrest, but the permeability of the blood-spinal cord barrier (BSCB) to both materials was unchanged with time. We conclude that selective weight loss occurs in the brain after cardiac arrest with the integrity of the BBB and BSCB remaining intact to serum proteins and minimal alteration in the blood to CNS transport of TNF.     

Effect of Diabetes Mellitus on the Permeability of the Blood–Brain Barrier to Insulin

Banks, W. A., J. B. Jaspan and A. J. Kastin. Effect of diabetes mellitus on the permeability of the blood–brain barrier to insulin. Peptides 18(10) 1577–1584, 1997.—Insulin derived from the peripheral circulation has been shown to exert various effects on the brain due to its ability to cross the blood–brain barrier (BBB). The relation between diabetes mellitus and insulin has been extensively studied for peripheral tissues but not for central nervous system tissues. We examined the effects that streptozotocin- or alloxan-induced diabetes have on the transport of insulin across the murine BBB. We used multiple-time regression analysis to measure the unidirectional influx rate constant (K_i) and vascular association (V_i) of intravenously injected, radioactively labeled human insulin (I-Ins). Treatment with streptozotocin induced an enhancement of both the K_i and V_i of I-Ins that correlated with the onset of diabetes. Brain perfusion showed that the enhanced uptake was not due to altered vascular space or levels of insulin in the serum. Alloxan enhanced K_i and V_i after 5 days but the early phase of diabetes was associated with a decreased K_i. Hyperglycemia induced by the intraperitoneal injection of glucose elevated the V_i but abolished the K_i. Furthermore, altered I-Ins uptake by brain was not associated with changes in brain or body weight. These results show that there is an increased uptake of I-Ins by the brain in the diabetic state that is not due to acute changes in the serum levels of glucose or insulin, altered vascular space, or catabolic events. Chronic changes in levels of glucose, insulin or other hormone or neuroendocrine agents are likely to underlie the altered rate of transport of insulin across the BBB of diabetic mice.     

Myo-inositol transport through the blood-brain barrier

The unidirectional transport of [³H]myo-inositol across cerebral capillaries, the anatomical locus of the blood-brain barrier, was measured using an in situ rat brain perfusion technique. Myo-inositol was transported across the blood-brain barrier by a low capacity, saturable system with a one-half saturation concentration of 0.1 mM. The permeability surface-area product was 6.2×10^–5S^–1 with a myo-inositol concentration of 0.02 mM in the perfusate. The myo-inositol stereoisomer scyllo-inositol but not (+)-chiro-inositol (both 1 mM) inhibited myo-inositol transfer through the blood-brain barrier. These observations provide evidence that myo-inositol is transferred through the blood-brain barrier by simple diffusion and a stereospecific, saturable transport system.