The possible role of astrocytes in the regulation of feeding has been overlooked. It is well-established that the endothelial cells constituting the blood-brain barrier transport leptin from blood to brain and that hypothalamic neurons respond to leptin to induce anorexic signaling. However, few studies have addressed the role of astrocytes in either leptin transport or cellular activation. We recently showed that the obese agouti viable yellow mouse has prominent astrocytic expression of the leptin receptor. In this study, we test the hypothesis that diet-induced obesity increases astrocytic leptin receptor expression and function in the hypothalamus. Double-labelling immunohistochemistry and confocal microscopic analysis showed that all astrocytes in the hypothalamus express leptin receptors. In adult obese mice, 2 months after being placed on a high-fat diet, there was a striking increase of leptin receptor (+) astrocytes, most prominent in the dorsomedial hypothalamus and arcuate nucleus. Agouti viable yellow mice with their adult-onset obesity showed similar changes, but the increase of leptin receptor (+) astrocytes was barely seen in ob/ob or db/db mice with their early-onset obesity and defective leptin systems. The marked leptin receptor protein expression in the astrocytes, shown with several antibodies against different receptor epitopes, was supported by RT-PCR detection of leptin receptor-a and -b mRNAs in primary hypothalamic astrocytes. Unexpectedly, the protein expression of GFAP, a marker of astrocytes, was also increased in adult-onset obesity. Real-time confocal imaging showed that leptin caused a robust increase of calcium signalling in primary astrocytes from the hypothalamus, confirming their functionality. The results indicate that metabolic changes in obese mice can rapidly alter leptin receptor expression and astrocytic activity, and that leptin receptor is responsible for leptin-induced calcium signalling in astrocytes. This novel and clinically relevant finding opens new avenues in astrocyte biology.

The blood-brain barrier (BBB) provides a vast interface for cytokines to affect CNS function. The BBB is a target for therapeutic intervention. It is essential, therefore, to understand how cytokines interact with each other at the level of the BBB and how secondary signals modulate CNS functions beyond the BBB. The interactions between cytokines and lipids, however, have not been fully addressed at the level of the BBB. Here, we summarize current understanding of the localization of cytokine receptors and transporters in specific membrane microdomains, particularly lipid rafts, on the luminal (apical) surface of the microvascular endothelial cells composing the BBB. We then illustrate the clinical context of cytokine effects on the BBB by neuroendocrine regulation and amplification of inflammatory signals. Two unusual aspects discussed are signaling crosstalk by different classes of cytokines and genetic regulation of drug efflux transporters. We also introduce a novel area of focus on how cytokines may act through nuclear hormone receptors to modulate efflux transporters and other targets. A specific example discussed is the ATP-binding cassette transporter-1 (ABCA-1) that regulates lipid metabolism. Overall, cytokine signaling at the level of the BBB is a crucial feature of the dynamic regulation that can rapidly change BBB function and affect brain health and disease.

The blood-brain barrier (BBB) is a large regulatory and exchange interface between the brain and peripheral circulation. We propose that changes of the BBB contribute to many pathophysiological processes in the brain of subjects with chronic sleep restriction (CSR). To achieve CSR that mimics a common pattern of human sleep loss, we quantified a new procedure of sleep disruption in mice by a week of consecutive sleep recording. We then tested the hypothesis that CSR compromises microvascular function. CSR not only diminished endothelial and inducible nitric oxide synthase, endothelin1, and glucose transporter expression in cerebral microvessels of the BBB, but it also decreased 2-deoxy-glucose uptake by the brain. The expression of several tight junction proteins also was decreased, whereas the level of cyclooxygenase-2 increased. This coincided with an increase of paracellular permeability of the BBB to the small tracers sodium fluorescein and biotin. CSR for 6 d was sufficient to impair BBB structure and function, although the increase of paracellular permeability returned to baseline after 24 h of recovery sleep. This merits attention not only in neuroscience research but also in public health policy and clinical practice.

FGF21 recently has been proposed as a missing link in the biology of fasting, raising the question of whether it directly reaches the brain. We used multiple time-regression analysis to quantify the influx rate of this polypeptide across the blood-brain barrier (BBB), size-exclusion chromatography to examine degradation, capillary depletion to differentiate entry into brain parenchyma from retention in the microvasculature, and measurement of efflux rate to determine a possible confounding effect on measurement of entry. FGF21 was 94% intact in serum and 75% in brain 10 min after intravenous bolus delivery. Its influx rate was 0.23+/-0.12 microl/g-min, nearly four times faster than that of the vascular marker albumin. At 10 min, about 0.5% of the administered FGF21 was present in a gram of brain tissue. Of this, 70% reached the parenchyma of the brain. Co-injection of excess FGF21 failed to inhibit the influx, showing a lack of saturation. Efflux, which occurred at the same rate as the bulk reabsorption of cerebrospinal fluid, also was not saturable. In summary, FGF21 shows significant, non-saturable, unidirectional influx across the BBB.

The agouti viable yellow (A vy) spontaneous mutation generates an unusual mouse phenotype of agouti-colored coat and adult-onset obesity with metabolic syndrome. Persistent production of agouti signaling protein in A vy mice antagonizes melanocortin receptors in the hypothalamus. To determine how this disruption of neuroendocrine circuits affects leptin transport across the blood-brain barrier (BBB), we measured leptin influx in A vy and B6 control mice after the development of obesity, hyperleptinemia, and increased adiposity. After iv bolus injection, (125)I-leptin crossed the BBB significantly faster in young (2 month old) B6 mice than in young A vy mice or in older (8 month old) mice of either strain. This difference was not observed by in situ brain perfusion studies, indicating the cause being circulating factors, such as elevated leptin levels or soluble receptors. Thus, A vy mice showed peripheral leptin resistance. ObRa, the main transporting receptor for leptin at the BBB, showed no change in mRNA expression in the cerebral microvessels between the age-matched (2 month old) A vy and B6 mice. Higher ObRb mRNA was seen in the A vy microvasculature with unknown significance. Immunofluorescent staining unexpectedly revealed that many of the ObR(+) cells were astrocytes and that the A vy mice showed significantly more ObR(+) astrocytes in the hypothalamus than the B6 mice. Although leptin permeation from the circulation was slower in the A vy mice, the increased ObR expression in astrocytes and increased ObRb mRNA in microvessels suggest the possibility of heightened central nervous system sensitivity to circulating leptin.

Background/Aims: Acute phase C-reactive protein (CRP), elevated in obesity and inflammation, is a major binding protein for leptin. It is thought that CRP contributes to leptin resistance by preventing leptin from crossing the blood-brain barrier (BBB). Here we determined how CRP interacts with the BBB and whether it deters leptin from reaching CNS targets. Methods: BBB permeability, compartmental distribution, tracer stability, and expression of tight junction protein and inflammatory marker were determined. Results: CRP was stable in blood, but did not permeate the BBB in trace amounts. However, it increased paracellular permeability at a higher dose. Agouti viable (A^vy) mice with adult-onset obesity show higher CRP entry into the brain. CRP did not permeate hCMEC/D3 cells nor change zona occludin-1 or cyclooxygenase-2 expression. An intermediate dose of CRP had no effect on leptin transport across the BBB after co-treatment. Thus, acute interactions between CRP and leptin at the BBB level were negligible and did not explain the leptin resistance seen in obesity. Conclusions: The interactions of CRP and the BBB are a two-phase process, with increased paracellular permeability at a high dose that enables its entry into the CNS and serves to induce reactive gliosis and impair CNS function.

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Interleukin-15 (IL-15) is a cytokine produced in the normal brain that acts on its specific receptor IL-15Rα and co-receptors IL-2Rβ and IL-2Rγ in neuronal cells. The functions of the cerebral IL-15 system, however, are not yet clear. To test the hypothesis that IL-15Rα regulates metabolic activity and body temperature, we quantified the specific metabolic phenotype of IL-15Rα knockout mice. These normal-appearing mice were leaner with lower fat composition. During the entire circadian cycle, the knockout mice had a significantly higher acrophase in locomotor activity and heat dissipation. During the light phase, there was significantly greater food intake, oxygen consumption, and carbon dioxide production. The difference in the dark and light phases suggests that IL-15Rα participates in circadian rhythm regulation. The higher oxygen consumption in the light phase indicates adaptive thermogenesis in the knockout mice. The body temperature of the receptor knockout mice was significantly higher than the control in the light phase, and this was mainly caused by a large difference occurring between 0600 and 0900 h. In addition to the metabolic chamber studies and circadian rhythm analyses, qPCR of hypothalamic homogenates indicated higher mRNA expression of orexin and transient receptor potential vanilloid 4 cation channels. Consistent with a direct role of IL-15Rα in the hypothalamus, IL-15 treatment of the wild-type mice induced c-Fos expression in the preoptic area. We conclude that activation of hypothalamic neurons by IL-15 in mice contributes to thermoregulation and modifies the metabolic phenotype.