Hypothalamic Glial Cells Research Articles

Cloning and Molecular Characterization of HSL and Its Expression Pattern in HPG Axis and Testis during Different Stages in Bactrian Camel.

Hormone-sensitive lipase (HSL) is a key enzyme in animal fat metabolism and is involved in the rate-limiting step of catalyzing the decomposition of fat and cholesterol. It also plays an important regulatory role in maintaining seminiferous epithelial structure, androgen synthesis and primordial germ cell differentiation. We previously reported that HSL is involved the synthesis of steroids in Bactrian camels, although it is unclear what role it plays in testicular development. The present study was conducted to characterize the biological function and expression pattern of the HSL gene in the hypothalamic pituitary gonadal (HPG) axis and the development of testis in Bactrian camels. We analyzed cloning of the cDNA sequence of the HSL gene of Bactrian camels by RT-PCR, as well as the structural features of HSL proteins, using bioinformatics software, such as ProtParam, TMHMM, Signal P 4.1, SOPMA and MEGA 7.0. We used qRT-PCR, Western blotting and immunofluorescence staining to clarify the expression pattern of HSL in the HPG axis and testis of two-week-old (2W), two-year-old (2Y), four-year-old (4Y) and six-year-old (6Y) Bactrian camels. According to sequence analysis, the coding sequence (CDS) region of the HSL gene is 648 bp in length and encodes 204 amino acids. According to bioinformatics analysis, the nucleotide and amino acid sequence of Bactrian camel HSL are most similar to those of Camelus pacos and Camelus dromedarius, with the lowest sequence similarity with Mus musculus. In adult Bactrian camel HPG axis tissues, both HSL mRNA and protein expression were significantly higher in the testis than in other tissues (hypothalamus, pituitary and pineal tissues) (p < 0.05). The expression of mRNA in the testis increased with age and was the highest in six-year-old testis (p < 0.01). The protein expression levels of HSL in 2Y and 6Y testis were clearly higher than in 2W and 4Y testis tissues (p < 0.01). Immunofluorescence results indicate that the HSL protein was mainly localized in the germ cells, Sertoli cells and Leydig cells from Bactrian camel testis, and strong positive signals were detected in epididymal epithelial cells, basal cells, spermatocytes and smooth muscle cells, with partially expression in hypothalamic glial cells, pituitary suspensory cells and pineal cells. According to the results of gene ontology (GO) analysis enrichment, HSL indirectly regulates the anabolism of steroid hormones through interactions with various targets. Therefore, we conclude that the HSL gene may be associated with the development and reproduction of Bactrian camels in different stages of maturity, and these results will contribute to further understanding of the regulatory mechanisms of HSL in Bactrian camel reproduction.

1394-P: Peri-Islet Glial-Cell Expansion Occurs in Response to High-Fat Diet-Induced Obesity and Hyperglycemia

Peri-islet glial cells (PIGCs) are positioned to control neuronal-endocrine-vascular cell communication within the islet, but their role in islet cell function is not known. Hypothalamic glial cells become reactive in response to high fat diet (HFD) feeding and are implicated in the pathogenesis of obesity and type 2 diabetes. We hypothesize that metabolic stress such as diet-induced obesity and hyperglycemia promote the expansion of PIGCs, similar to what has been observed in the central nervous system. To test this hypothesis, we used the glial cell marker, glial fibrillary acidic protein (GFAP) , to identify PIGCs and compared GFAP immunoreactivity in pancreas from the following groups. First, pancreas was harvested from HFD- or chow fed control C57BL/6J male mice after 2 days, 2 weeks or 8 weeks on diet (n=6-8/group) . Second, pancreas was taken from 12-wk old db/db male mice and their control littermates with or without empagliflozin (EMPA) treatment (20mg/kg/d for 6 weeks; n=4-7/group) . Islet PIGC area was quantified as GFAP-positive area/total islet area x100%. Mann-Whitney test and one-way ANOVA were used for data analysis. We found that HFD feeding increased mouse pancreatic peri-islet GFAP expression relative to chow controls at 2 days (HFD 1.4±0.2%, chow 0.8±0.1%; p=0.02) , 2 weeks (HFD 3.5±0.4%, chow 1.2±0.1%; p&amp;lt;0.001) and 8 weeks (HFD 5.4±0.3%, chow 1.8±0.2%; p=0.002) in association with body weight gain and impaired glucose tolerance. Similar results were observed in obese and hyperglycemic db/db mouse vs. control littermates (db/db 5.0±1.4%, control 1.2±0.4%; p=0.004) . There was a trend for EMPA treatment to reduce islet PIGC area in the db/db mice (EMPA 2.0±0.5, VEH 5.0±1.4%, p=0.08) . In conclusion, these findings indicate that islet gliosis is observed in two obese and hyperglycemic mouse models and raise the possibility that pancreatic ‘gliosis’ may have a relevant role in islet-cell function regulation under metabolic challenge. Disclosure J.Niu: None. J.B.Moss: None. W.Rosario: Employee; Merck &amp; Co., Inc. R.Arumugam: None. D.J.Hackney: None. R.L.Hull-meichle: Research Support; Casma Therapeutics. J.Tong: None. Funding National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases (R01DK097550) ;Department of Veterans Affairs (IBX004063)

Metabolic changes in hypothalamic glial cells under pathological conditions of cancer cachexia

Metabolic changes in hypothalamic glial cells under pathological conditions of cancer cachexia

Normal aging induces PD-1-enriched exhausted microglia and A1-like reactive astrocytes in the hypothalamus

Hypothalamic aging is considered to be critical for systemic aging, and the accumulation of “exhausted glial cells” in the hypothalamus may contribute to brain dysfunction. In this study, we used normal aging mice and investigated aging-specific transcriptional identities of microglia and astrocytes in the hypothalamus. We confirmed that normal aging promoted anxiety, induced impairment of motor coordination and reduced physical strength of muscle in mice. To investigate the senescence of hypothalamic glial cells, we isolated CD11b-positive microglia and ACSA-2-positive astrocytes from the hypothalamus of aged mice using magnetic-activated cell sorting (MACS). The mRNA level of p16INK4A was dramatically increased in the hypothalamic microglia of aged mice compared to young mice. Furthermore, the expression of programmed cell death 1 (PD-1) as well as A1-like astrocyte mediators in the hypothalamic microglia was dramatically induced by aging, indicating that normal aging may produce PD-1-enriched “exhausted microglia” in the hypothalamus. Furthermore, neuroinflammatory A1-like reactive astrocytes with a p16INK4A-positive senescent state were predominantly detected in the hypothalamus of aged mice. Exhausted microglia were also detected in the prefrontal cortex of aged mice, whereas astrocytic neuroinflammation was milder than that observed in the hypothalamus, even with p16INK4A-positive senescence. These results suggest that the production of PD-1-enriched exhausted and senescent microglia and neuroinflammatory A1-like reactive astrocytes in the hypothalamus may partly contribute to aging-related emotional and physical dyscoordination.

Transcriptomic analysis links diverse hypothalamic cell types to fibroblast growth factor 1-induced sustained diabetes remission

In rodent models of type 2 diabetes (T2D), sustained remission of hyperglycemia can be induced by a single intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1), and the mediobasal hypothalamus (MBH) was recently implicated as the brain area responsible for this effect. To better understand the cellular response to FGF1 in the MBH, we sequenced >79,000 single-cell transcriptomes from the hypothalamus of diabetic Lepob/ob mice obtained on Days 1 and 5 after icv injection of either FGF1 or vehicle. A wide range of transcriptional responses to FGF1 was observed across diverse hypothalamic cell types, with glial cell types responding much more robustly than neurons at both time points. Tanycytes and ependymal cells were the most FGF1-responsive cell type at Day 1, but astrocytes and oligodendrocyte lineage cells subsequently became more responsive. Based on histochemical and ultrastructural evidence of enhanced cell-cell interactions between astrocytes and Agrp neurons (key components of the melanocortin system), we performed a series of studies showing that intact melanocortin signaling is required for the sustained antidiabetic action of FGF1. These data collectively suggest that hypothalamic glial cells are leading targets for the effects of FGF1 and that sustained diabetes remission is dependent on intact melanocortin signaling.

Hypothalamic glial cells isolated by MACS reveal that microglia and astrocytes induce hypothalamic inflammation via different processes under high-fat diet conditions

Glial cells can mediate hypothalamic inflammatory processes induced in response to a high-fat diet (HFD). We used magnetic-activated cell sorting (MACS) to isolate microglia and astrocytes from hypothalamus of mice fed HFD and examined changes in expression of inflammation-related cytokines and markers related to glial cell activation status.Hypothalamus from male C57BL6 mice fed a chow diet (chow) or HFD for 1, 3, or 28 days were collected and microglia and astrocytes were isolated by MACS. After confirming cell viability by fluorescence activated cell sorting, mRNA expression levels of inflammation-related cytokines and markers of glial cell activation status were examined by qRT-PCR, which revealed that both glial cell types isolated by MACS retained specificity.On day 3 of HFD, both CD86 and TNFα mRNA expression was significantly increased in microglia relative to the chow group. In astrocytes, TNFα mRNA expression levels were similar between the chow and HFD groups on day 3, but anti-inflammatory cytokine IL-10 levels were significantly increased. On day 7 of HFD, TNFα expression in microglia decreased to levels comparable to the chow group while that in astrocytes remained unchanged. On day 28 of HFD, TNFα levels were significantly increased in both microglia and astrocytes, which had increased mRNA expression of CD86 and MAO-B, respectively. For both glial cell types, results for TNFα expression assessed by RT-PCR and immunohistochemical analysis were similar.These results indicate that the role of microglia and astrocytes in hypothalamic inflammation under HFD conditions changed with time and these changes were accompanied by changes in the activation status of glial cells. Our data suggest that early after initiating HFD, hypothalamic astrocytes suppress diet-induced inflammation at least in part by secreting IL-10, whereas continued HFD feeding impairs this suppressive function such that both microglia and astrocytes promote hypothalamic inflammation.

Tanycytic TSPO inhibition induces lipophagy to regulate lipid metabolism and improve energy balance

Hypothalamic glial cells named tanycytes, which line the 3rd ventricle (3V), are components of the hypothalamic network that regulates a diverse array of metabolic functions for energy homeostasis....

Tanycytic TSPO inhibition induces lipophagy to regulate lipid metabolism and improve energy balance

ABSTRACT Hypothalamic glial cells named tanycytes, which line the 3rd ventricle (3V), are components of the hypothalamic network that regulates a diverse array of metabolic functions for energy homeostasis. Herein, we report that TSPO (translocator protein), an outer mitochondrial protein, is highly enriched in tanycytes and regulates homeostatic responses to nutrient excess as a potential target for an effective intervention in obesity. Administration of a TSPO ligand, PK11195, into the 3V, and tanycyte-specific deletion of Tspo reduced food intake and elevated energy expenditure, leading to negative energy balance in a high-fat diet challenge. Ablation of tanycytic Tspo elicited AMPK-dependent lipophagy, breaking down lipid droplets into free fatty acids, thereby elevating ATP in a lipid stimulus. Our findings suggest that tanycytic TSPO affects systemic energy balance through macroautophagy/autophagy-regulated lipid metabolism, and highlight the physiological significance of TSPO in hypothalamic lipid sensing and bioenergetics in response to overnutrition. Abbreviations 3V: 3rd ventricle; ACAC: acetyl-Coenzyme A carboxylase; AGRP: agouti related neuropeptide; AIF1/IBA1: allograft inflammatory factor 1; AMPK: AMP-activated protein kinase; ARC: arcuate nucleus; Atg: autophagy related; Bafilo: bafilomycin A1; CAMKK2: calcium/calmodulin-dependent protein kinase kinase 2, beta; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CNS: central nervous system; COX4I1: cytochrome c oxidase subunit 4I1; FFA: free fatty acid; GFAP: glial fibrillary acidic protein; HFD: high-fat diet; ICV: intracerebroventricular; LAMP2: lysosomal-associated membrane protein 2; LD: lipid droplet; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MBH: mediobasal hypothalamus; ME: median eminence; MEF: mouse embryonic fibroblast; NCD: normal chow diet; NEFM/NFM: neurofilament medium; NPY: neuropeptide Y; OL: oleic acid; POMC: pro-opiomelanocortin-alpha; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; Rax: retina and anterior neural fold homeobox; RBFOX3/NeuN: RNA binding protein, fox-1 homolog (C. elegans) 3; RER: respiratory exchange ratio; siRNA: small interfering RNA; SQSTM1: sequestosome 1; TG: triglyceride; TSPO: translocator protein; ULK1: unc-51 like kinase 1; VCO2: carbon dioxide production; VMH: ventromedial hypothalamus; VO2: oxygen consumption

Adenosine A1 receptors modulate the Na+-Hypertonicity induced glutamate release in hypothalamic glial cells

Glutamate release in response to a hypertonic stimulus is a well described phenomenon in the hypothalamus. Evidence suggests that hypothalamic glial cells release glutamate into the extracellular environment in hypertonic conditions. In the current study, we described autocrine regulation of adenosine on glutamate release induced by Na+hypertonicity in hypothalamic glial cell cultures. We showed that glial cells cultured from the cerebral cortex did not release glutamate or adenosine under hypertonic conditions. The findings suggest that the hypothalamus has specialized glial cells, which are responsive to osmotic variations. Stimulation or inhibition of adenosine A1 receptors modulates extracellular glutamate levels in hypothalamic glial cell cultures under hypertonic stimulation. Our results extend previous observations regarding the role of glial cells in the control of hypothalamic physiology. They further demonstrate for the first time that hypothalamic glial cells regulate Na+-hypertonicity-induced glutamate release by activation of adenosine A1 receptors via adenosine release.

Hypothalamic inflammation and energy balance deregulations: focus on chemokines.

The hypothalamus is a key brain region in the regulation of energy balance. It especially controls food intake and both energy storage and expenditure through integration of humoral, neural and nutrient-related signals and cues. Hypothalamic neurons and glial cells act jointly to orchestrate, both spatially and temporally, regulated metabolic functions of the hypothalamus. Thus, the existence of a causal link between hypothalamic inflammation and deregulations of feeding behavior, such as involuntary weight-loss or obesity, has been suggested. Among the inflammatory mediators that could induce deregulations of hypothalamic control of the energy balance, chemokines represent interesting candidates. Indeed, chemokines, primarily known for their chemoattractant role of immune cells to the inflamed site, have also been suggested capable of neuromodulation. Thus, chemokines could disrupt cellular activity together with synthesis and/or secretion of multiple neurotransmitters/mediators that are involved in the maintenance of energy balance. Here, we relate, on one hand, recent results showing the primary role of the central chemokinergic signaling CCL2/CCR2 for metabolic and behavioral adaptation to high-grade inflammation, especially loss of appetite and weight, through its activity on hypothalamic neurons producing the orexigenic peptide Melanin-Concentrating Hormone (MCH) and, on the other hand, results that suggest that chemokines could also deregulate hypothalamic neuropeptidergic circuits to induce an opposite phenotype and eventually participate in the onset/development of obesity. In more details, we will emphasize a study recently showing, in a model of high-grade acute inflammation of LPS injection in mice, that central CCL2/CCR2 signaling is of primary importance for several aspects explaining weight loss associated with inflammation: after LPS injection, animals lose weight, reduce their food intake, increase their fat oxidation (thus energy consumption from fat storage)...These inflammation-induced metabolic and behavioral changes are reduced when central CCR2 signaling is disrupted either pharmacologically (by a specific inhibitor of CCR2) or genetically (in mice deficient for CCR2). This underlines the importance of this signaling in inflammation-related weight loss. We further determined that the LPS-induced and CCR2-mediated weight loss depends on the direct effect of CCR2 activation on MCH neurons activity. Indeed, the MCH neurons express CCR2, and the application of CCL2 on brain slices revealed that activation of CCR2 actually depolarizes MCH neurons and induces delays and/or failures of action potential emission. Furthermore, CCL2 is able to reduce KCl-evoked MCH secretion from hypothalamic explants. Taken together, these results demonstrate the role of the central CCL2/CCR2 signaling in metabolic and behavioral adaptation to inflammation. On the other hand, this first description of how the chemokinergic system can actually modulate the activity of the hypothalamic regulation of energy balance, but also some less advanced studies and some unpublished data, suggest that some other chemokines, such as CCL5, could participate in the development of the opposite phenotype, that is to say obesity.

The role of tanycytes in hypothalamic glucosensing.

Tanycytes are elongated hypothalamic glial cells that cover the basal walls of the third ventricle; their apical regions contact the cerebrospinal fluid (CSF), and their processes reach hypothalamic neuronal nuclei that control the energy status of an organism. These nuclei maintain the balance between energy expenditure and intake, integrating several peripheral signals and triggering cellular responses that modify the feeding behaviour and peripheral glucose homeostasis. One of the most important and well-studied signals that control this process is glucose; however, the mechanism by which this molecule is sensed remains unknown. We along with others have proposed that tanycytes play a key role in this process, transducing changes in CSF glucose concentration to the neurons that control energy status. Recent studies have demonstrated the expression and function of monocarboxylate transporters and canonical pancreatic β cell glucose sensing molecules, including glucose transporter 2 and glucokinase, in tanycytes. These and other data, which will be discussed in this review, suggest that hypothalamic glucosensing is mediated through a metabolic interaction between tanycytes and neurons through lactate. This article will summarize the recent evidence that supports the importance of tanycytes in hypothalamic glucosensing, and discuss the possible mechanisms involved in this process. Finally, it is important to highlight that a detailed analysis of this mechanism could represent an opportunity to understand the evolution of associated pathologies, including diabetes and obesity, and identify new candidates for therapeutic intervention.

MCT2 Expression and Lactate Influx in Anorexigenic and Orexigenic Neurons of the Arcuate Nucleus

Hypothalamic neurons of the arcuate nucleus control food intake, releasing orexigenic and anorexigenic neuropeptides in response to changes in glucose concentration. Several studies have suggested that the glucosensing mechanism is governed by a metabolic interaction between neurons and glial cells via lactate flux through monocarboxylate transporters (MCTs). Hypothalamic glial cells (tanycytes) release lactate through MCT1 and MCT4; however, similar analyses in neuroendocrine neurons have yet to be undertaken. Using primary rat hypothalamic cell cultures and fluorimetric assays, lactate incorporation was detected. Furthermore, the expression and function of MCT2 was demonstrated in the hypothalamic neuronal cell line, GT1-7, using kinetic and inhibition assays. Moreover, MCT2 expression and localization in the Sprague Dawley rat hypothalamus was analyzed using RT-PCR, in situ hybridization and Western blot analyses. Confocal immunohistochemistry analyses revealed MCT2 localization in neuronal but not glial cells. Moreover, MCT2 was localized to ∼90% of orexigenic and ∼60% of anorexigenic neurons as determined by immunolocalization analysis of AgRP and POMC with MCT2-positives neurons. Thus, MCT2 distribution coupled with lactate uptake by hypothalamic neurons suggests that hypothalamic neurons control food intake using lactate to reflect changes in glucose levels.

Glucose Transporter-1 in the Hypothalamic Glial Cells Mediates Glucose Sensing to Regulate Glucose Production In Vivo

OBJECTIVECirculating glucose inhibits glucose production in normal rodents and humans, but this glucose effectiveness is disrupted in diabetes due partly to sustained hyperglycemia. We hypothesize that hyperglycemia in diabetes impairs hypothalamic glucose sensing to lower glucose production, and changes of glucose transporter-1 (GLUT1) in the hypothalamic glial cells are responsible for the deleterious effects of hyperglycemia in vivo.RESEARCH DESIGN AND METHODSWe tested hypothalamic glucose effectiveness to increase hypothalamic glucose concentration and lower glucose production in rats induced with streptozotocin (STZ) uncontrolled diabetes, STZ and phlorizin, and whole-body and hypothalamic sustained hyperglycemia. We next assessed the content of glial GLUT1 in the hypothalamus, generated an adenovirus expressing GLUT1 driven by a glial fibrillary acidic protein (GFAP) promoter (Ad-GFAP-GLUT1), and injected Ad-GFAP-GLUT1 into the hypothalamus of rats induced with hyperglycemia. Pancreatic euglycemic clamp and tracer-dilution methodologies were used to assess changes in glucose kinetics in vivo.RESULTSSustained hyperglycemia, as seen in the early onset of STZ-induced diabetes, disrupted hypothalamic glucose sensing to increase hypothalamic glucose concentration and lower glucose production in association with reduced GLUT1 levels in the hypothalamic glial cells of rats in vivo. Overexpression of hypothalamic glial GLUT1 in STZ-induced rats with reduced GLUT1 acutely normalized plasma glucose levels and in rats with selectively induced hypothalamic hyperglycemia restored hypothalamic glucose effectiveness.CONCLUSIONSSustained hyperglycemia impairs hypothalamic glucose sensing to lower glucose production through changes in hypothalamic glial GLUT1, and these data highlight the critical role of hypothalamic glial GLUT1 in mediating glucose sensing to regulate glucose production.

MCT Expression and Lactate Influx/Efflux in Tanycytes Involved in Glia-Neuron Metabolic Interaction

Metabolic interaction via lactate between glial cells and neurons has been proposed as one of the mechanisms involved in hypothalamic glucosensing. We have postulated that hypothalamic glial cells, also known as tanycytes, produce lactate by glycolytic metabolism of glucose. Transfer of lactate to neighboring neurons stimulates ATP synthesis and thus contributes to their activation. Because destruction of third ventricle (III-V) tanycytes is sufficient to alter blood glucose levels and food intake in rats, it is hypothesized that tanycytes are involved in the hypothalamic glucose sensing mechanism. Here, we demonstrate the presence and function of monocarboxylate transporters (MCTs) in tanycytes. Specifically, MCT1 and MCT4 expression as well as their distribution were analyzed in Sprague Dawley rat brain, and we demonstrate that both transporters are expressed in tanycytes. Using primary tanycyte cultures, kinetic analyses and sensitivity to inhibitors were undertaken to confirm that MCT1 and MCT4 were functional for lactate influx. Additionally, physiological concentrations of glucose induced lactate efflux in cultured tanycytes, which was inhibited by classical MCT inhibitors. Because the expression of both MCT1 and MCT4 has been linked to lactate efflux, we propose that tanycytes participate in glucose sensing based on a metabolic interaction with neurons of the arcuate nucleus, which are stimulated by lactate released from MCT1 and MCT4-expressing tanycytes.

Glial Glucokinase Expression in Adult and Post-Natal Development of the Hypothalamic Region

It has recently been proposed that hypothalamic glial cells sense glucose levels and release lactate as a signal to activate adjacent neurons. GK (glucokinase), the hexokinase involved in glucose sensing in pancreatic β-cells, is also expressed in the hypothalamus. However, it has not been clearly determined if glial and/or neuronal cells express this protein. Interestingly, tanycytes, the glia that cover the ventricular walls of the hypothalamus, are in contact with CSF (cerebrospinal fluid), the capillaries of the arcuate nucleus and adjacent neurons; this would be expected for a system that can detect and communicate changes in glucose concentration. Here, we demonstrated by Western-blot analysis, QRT–PCR [quantitative RT–PCR (reverse transcription–PCR)] and in situ hybridization that GK is expressed in tanycytes. Confocal microscopy and immunoultrastructural analysis revealed that GK is localized in the nucleus and cytoplasm of β1-tanycytes. Furthermore, GK expression increased in these cells during the second week of post-natal development. Based on this evidence, we propose that tanycytes mediate, at least in part, the mechanism by which the hypothalamus detects changes in glucose concentrations.

Sodium vitamin C cotransporter SVCT2 is expressed in hypothalamic glial cells

Kinetic analysis of vitamin C uptake demonstrated that different specialized cells take up ascorbic acid through sodium-vitamin C cotransporters. Recently, two different isoforms of sodium-vitamin C cotransporters (SVCT1/SLC23A1 and SVCT2/SLC23A2) have been cloned. SVCT2 was detected mainly in choroidal plexus cells and neurons; however, there is no evidence of SVCT2 expression in glial and endothelial cells of the brain. Certain brain locations, including the hippocampus and hypothalamus, consistently show higher ascorbic acid values compared with other structures within the central nervous system. However, molecular and kinetic analysis addressing the expression of SVCT transporters in cells isolated from these specific areas of the brain had not been done. The hypothalamic glial cells, or tanycytes, are specialized ependymal cells that bridge the cerebrospinal fluid with different neurons of the region. Our hypothesis postulates that SVCT2 is expressed selectively in tanycytes, where it is involved in the uptake of the reduced form of vitamin C (ascorbic acid), thereby concentrating this vitamin in the hypothalamic area. In situ hybridization and optic and ultrastructural immunocytochemistry showed that the transporter SVCT2 is highly expressed in the apical membranes of mouse hypothalamic tanycytes. A newly developed primary culture of mouse hypothalamic tanycytes was used to confirm the expression and function of the SVCT2 isoform in these cells. The results demonstrate that tanycytes express a high-affinity transporter for vitamin C. Thus, the vitamin C uptake mechanisms present in the hypothalamic glial cells may perform a neuroprotective role concentrating vitamin C in this specific area of the brain.

Hypothalamic ependymal-glial cells express the glucose transporter GLUT2, a protein involved in glucose sensing.

The GLUT2 glucose transporter and the K-ATP-sensitive potassium channels have been implicated as an integral part of the glucose-sensing mechanism in the pancreatic islet beta cells. The expression of GLUT2 and K-ATP channels in the hypothalamic region suggest that they are also involved in a sensing mechanism in this area. The hypothalamic glial cells, known as tanycytes alpha and beta, are specialized ependymal cells that bridge the cerebrospinal fluid and the portal blood of the median eminence. We used immunocytochemistry, in situ hybridization and transport analyses to demonstrate the glucose transporters expressed in tanycytes. Confocal microscopy using specific antibodies against GLUT1 and GLUT2 indicated that both transporters are expressed in alpha and beta tanycytes. In addition, primary cultures of mouse hypothalamic tanycytes were found to express both GLUT1 and GLUT2 transporters. Transport studies, including 2-deoxy-glucose and fructose uptake in the presence or absence of inhibitors, indicated that these transporters are functional in cultured tanycytes. Finally, our analyses indicated that tanycytes express the K-ATP channel subunit Kir6.1 in vitro. As the expression of GLUT2 and K-ATP channel is linked to glucose-sensing mechanisms in pancreatic beta cells, we postulate that tanycytes may be responsible, at least in part, for a mechanism that allows the hypothalamus to detect changes in glucose concentrations.

Poster Sessions CP10: Blood–Brain Barrier. Expression of vitamin C transporter SVCT2 in hypothalamic glial cells. An immunohistochemical and kinetic analysis

Kinetic analysis of vitamin C uptake has demonstrated that specialized cells take up ascorbic acid (AA), the reduced form of vitamin C, through sodium‐AA cotransporters. Recently, two different isoforms of sodium‐vitamin C cotransporters (SVCT 1, 2) that mediate high affinity Na+‐dependent l‐ascorbic acid have been cloned. SVCT2 was detected mainly in choroid plexus cells and neurons, however, there are no evidences of SVCT2 expression in glial cells. High concentrations of vitamin C has been demonstrated in brain hypothalamic area. The hypothalamic glial cells, known as alpha and beta tanycytes, are specialized ependymal cells that bridge the cerebrospinal fluid and the portal blood of the median eminence. Our hypothesis postulates that tanycytes take up reduced vitamin C from the portal blood and cerebrospinal fluid generating an high concentration of this vitamin in brain hypothalamic area. In situ immunohistochemical analyses demonstrated that SVCT2 transporter is selectively expressed in apical region of tanycytes. A newly developed primary culture of mouse hypothalamic tanycytes was used to confirm the expression and function of SVCT2 isoform in these cells. Reduced vitamin C uptake was temperature and sodium dependent. Kinetic analysis showed an apparent Km of 20 μm and a Vmax of 45 pmol/min per million cells for the transport of ascorbic acid. The expression of SVCT2 was confirmed by immunoblots and RT–PCR. Tanycytes may perform a neuroprotective role concentrating the vitamin C in the hypothalamic area.Acknowledgements: Supported by Grands FONDECYT 1010843 and DIUC‐GIA 201.034.006‐1.4 from Concepción University.

Hypothalamic ependymal cells express the glucose transporter GLUT2, a protein involved in glucose sensing

The GLUT2 glucose transporter is expressed in pancreatic islet β cells participating in glucose sensing mechanisms. In addition to glucose, GLUT2 is required in the transport of fructuose. In situ hybridization analyses of GLUT2 gene expression suggested that this transporter is present in hypothalamic regions responsible for glucose sensing. The hypothalamic glial cells, known as α and β tanycytes, are specialized ependymal cells that bridge the CSF and the blood. We used immunocytochemistry and transport analyses to demonstrate that GLUT1 and GLUT2 are expressed in tanycytes and are directly involved in the uptake of glucose and fructose. We also demonstrated that GLUT1 expression is elevated in β‐1 tanycytes, as compared to neurons and astrocytes. In addition, confocal microscopy analysis using anti‐GLUT2 indicated that GLUT2 was specifically expressed in β‐1 and β‐2 tanycytes. A newly developed primary culture of mouse hypothalamic tanycytes was found to express both GLUT1 and GLUT2. Analyses of 2‐deoxy‐d‐glucose and fructose uptake in cultured tanycytes revealed the presence of two transporters with transport Kms and sensitivity to inhibitors consistent with the functional properties of GLUT1 and GLUT2. Therefore, we conclude that mouse hypothalamic tanycytes express functional GLUT1 and GLUT2. Because the expression of GLUT2 is linked to glucose sensing mechanisms, we postulate that glial cells participate, at least in part, in glucose sensing mechanisms.Acknowledgements: Supported by Grands FONDECYT 1010843 and DIUC‐GIA 201.034.006–1.4 from Concepción University.

Neuronal vs. glial somatostatin in the hypothalamus: a cell culture study of the ontogenesis of cellular location, content and release

Somatostatin (SRIH) is an ubiquitous peptide subserving functions as a hypothalamic-hypophysial regulatory peptide and as a neuromodulator in the CNS, as well as various roules in the periphery. Based on our earlier observations that the ability of primary cultures of foetal rat hypothalamic cells to secrete SRIH developed distinct differences depending on whether they were maintained in a serum-supplemented medium (SSM) or a defined, serum free medium (DM), coupled with recent reports that glia in several brain regions may express neuropeptides, we have investigated the cellular location of SRIH in these cultures. Using immunocytochemical techniques (ICC) for the simultaneous location of SRIH and glial fibrillary acidic protein (GFAP) we found that SRIH-like immunoreactivity (SRIH-LI) was present in a significat number of hypothalamic glial cells around the time of birth. In the presence of serum, this expression persists and even by day 9 in culture a dense and intense immunofluorescent SRIH-LI signal was observed in GFAP positive cells. However, in DM glial expression is switched off rapidly (although GFAP positive cells are still present) until SRIH-LI eventually occurs at a much reduced abundance in GFAP negative neuronal cell types. In addition, the SRIH content of SSM cultures was three-fold greater than that from SSM cultures. Thus our results indicate that the mechanisms for SRIH-LI release from astrocytes appears to be distinctly different to that from neuronal-type cells. We propose that glial cell expression of SRIH-LI may represent a developmentally important phenomenon in the hypothalamus, e.g. serving a trophic role during critical times of development, and that its expression in glia may be stimulated by factors present in serum. Consequently, an understanding of factors which regulate SRIH expression and release from glia is likely to be importance to our understanding of brain development, injury, repair and diseases of the CNS.