Proteome analysis and conditional deletion of the EAAT2 glutamate transporter provide evidence against a role of EAAT2 in pancreatic insulin secretion in mice.

Islet function is incompletely understood in part because key steps in glutamate handling remain undetermined. The glutamate (excitatory amino acid) transporter 2 (EAAT2; Slc1a2) has been hypothesized to (a) provide islet cells with glutamate, (b) protect islet cells against high extracellular glutamate concentrations, (c) mediate glutamate release, or (d) control the pH inside insulin secretory granules. Here we floxed the EAAT2 gene to produce the first conditional EAAT2 knock-out mice. Crossing with Nestin-cyclization recombinase (Cre) eliminated EAAT2 from the brain, resulting in epilepsy and premature death, confirming the importance of EAAT2 for brain function and validating the genetic construction. Crossing with insulin-Cre lines (RIP-Cre and IPF1-Cre) to obtain pancreas-selective deletion did not appear to affect survival, growth, glucose tolerance, or ß-cell number. We found (using TaqMan RT-PCR, immunoblotting, immunocytochemistry, and proteome analysis) that the EAAT2 levels were too low to support any of the four hypothesized functions. The proteome analysis detected more than 7,000 islet proteins of which more than 100 were transporters. Although mitochondrial glutamate transporters and transporters for neutral amino acids were present at high levels, all other transporters with known ability to transport glutamate were strikingly absent. Glutamate-metabolizing enzymes were abundant. The level of glutamine synthetase was 2 orders of magnitude higher than that of glutaminase. Taken together this suggests that the uptake of glutamate by islets from the extracellular fluid is insignificant and that glutamate is intracellularly produced. Glutamine synthetase may be more important for islets than assumed previously.

Distribution of the glutamate transporters GLT-1 (SLC1A2) and GLAST (SLC1A3) in peripheral organs.

The glutamate transporters GLT-1 and GLAST are widely expressed in astrocytes in the brain where they fulfill important functions during glutamatergic neurotransmission. The present study examines their distribution in peripheral organs using in situ hybridization (ISH) and immunocytochemistry. GLAST was found to be more widely distributed than GLT-1. GLAST was expressed primarily in epithelial cells, cells of the macrophage-lineage, lymphocytes, fat cells, interstitial cells, and salivary gland acini. GLT-1 was primarily expressed in glandular tissue, including mammary gland, lacrimal gland, and ducts and acini in salivary glands, but also by perivenous hepatocytes and follicular dendritic cells in spleen and lymph nodes. The findings demonstrate that, although expressed by the same cells in the brain, these two glutamate transporters have different distribution patterns in peripheral tissues and that they fulfill glutamate transport functions apart from glutamatergic neurotransmission in these areas.

Cellular and subcellular mRNA localization of glutamate transporter isoforms GLT1a and GLT1b in rat brain by in situ hybridization.

GLT1, the predominant glutamate transporter of the forebrain, exists in two splice variant isoforms, i.e., GLT1a and GLT1b. Although GLT1 was originally detected only in astrocytes, we have recently demonstrated that GLT1a protein is expressed by neurons in the hippocampus as well. In the present study, the mRNA distribution patterns for the two isoforms were examined throughout the rat brain by using nonisotopic in situ hybridization and variant-specific RNA probes. Both isoforms were expressed in neuronal subgroups outside the hippocampus, such as in the cerebral cortex layer VI, or the neurons in the olfactory tubercle. As was the case in the hippocampus, GLT1a was the predominant transcript in neurons in these regions as well. Both GLT1 isoforms were widely expressed in astrocytes throughout the brain. GLT1a mRNA expression in astrocytes showed noticeable variation in labeling intensity in subregions of the hippocampus and other areas, whereas GLT1b expression in astrocytes was relatively homogeneous. On the subcellular level, GLT1a mRNA was expressed primarily in astrocyte processes, whereas GLT1b mRNA was more restricted to the astrocyte cell body. The two isoforms showed similar distributions in the subfornical organ and in tanycytes of the third ventricle. However, GLT1 expression in the pineal gland and the retina was due primarily to GLT1b, whereas GLT1a was more strongly expressed in Bergman glia in the cerebellum. These findings suggest that the expression of the two GLT1 isoforms is regulated by different mechanisms. Moreover, the function of the two isoforms may be subject to different regulatory processes.

Expression of the iron transporter ferroportin in synaptic vesicles and the blood-brain barrier.

Iron homeostasis in the mammalian brain is an important and poorly understood subject. Transferrin-bound iron enters the endothelial cells of the blood-brain barrier from the systemic circulation, and iron subsequently dissociates from transferrin to enter brain parenchyma by an unknown mechanism. In recent years, several iron transporters, including the iron importer DMT1 (Ireg1, MTP, DCT1) and the iron exporter ferroportin (SLC11A3, Ireg, MTP1) have been cloned and characterized. To better understand brain iron homeostasis, we have characterized the distribution of ferroportin, the presumed intestinal iron exporter, and have evaluated its potential role in regulation of iron homeostasis in the central nervous system. We discovered using in situ hybridization and immunohistochemistry that ferroportin is expressed in the endothelial cells of the blood-brain barrier, in neurons, oligodendrocytes, astrocytes, and the choroid plexus and ependymal cells. In addition, we discovered using techniques of immunoelectron microscopy and biochemical purification of synaptic vesicles that ferroportin is associated with synaptic vesicles. In the blood-brain barrier, it is likely that ferroportin serves as a molecular transporter of iron on the abluminal membrane of polarized endothelial cells. The role of ferroportin in synaptic vesicles is unknown, but its presence at that site may prove to be of great importance in neuronal iron toxicity. The widespread representation of ferroportin at sites such as the blood-brain barrier and synaptic vesicles raises the possibility that trafficking of elemental iron may be instrumental in the distribution of iron in the central nervous system.

The glutamate transporter GLT1a is expressed in excitatory axon terminals of mature hippocampal neurons.

GLT1 is the major glutamate transporter of the brain and has been thought to be expressed exclusively in astrocytes. Although excitatory axon terminals take up glutamate, the transporter responsible has not been identified. GLT1 is expressed in at least two forms varying in the C termini, GLT1a and GLT1b. GLT1 mRNA has been demonstrated in neurons, without associated protein. Recently, evidence has been presented, using specific C terminus-directed antibodies, that GLT1b protein is expressed in neurons in vivo. These data suggested that the GLT1 mRNA detected in neurons encodes GLT1b and also that GLT1b might be the elusive presynaptic transporter. To test these hypotheses, we used variant-specific probes directed to the 3'-untranslated regions for GLT1a and GLT1b to perform in situ hybridization in the hippocampus. Contrary to expectation, GLT1a mRNA was the more abundant form. To investigate further the expression of GLT1 in neurons in the hippocampus, antibodies raised against the C terminus of GLT1a and against the N terminus of GLT1, found to be specific by testing in GLT1 knock-out mice, were used for light microscopic and EM-ICC. GLT1a protein was detected in neurons, in 14-29% of axons in the hippocampus, depending on the region. Many of the labeled axons formed axo-spinous, asymmetric, and, thus, excitatory synapses. Labeling also occurred in some spines and dendrites. The antibody against the N terminus of GLT1 also produced labeling of neuronal processes. Thus, the originally cloned form of GLT1, GLT1a, is expressed as protein in neurons in the mature hippocampus and may contribute significantly to glutamate uptake into excitatory terminals.

Genetic ablations of iron regulatory proteins 1 and 2 reveal why iron regulatory protein 2 dominates iron homeostasis.

The two iron regulatory proteins IRP1 and IRP2 bind to transcripts of ferritin, transferrin receptor and other target genes to control the expression of iron metabolism proteins at the post-transcriptional level. Here we compare the effects of genetic ablation of IRP1 to IRP2 in mice. IRP1-/- mice misregulate iron metabolism only in the kidney and brown fat, two tissues in which the endogenous expression level of IRP1 greatly exceeds that of IRP2, whereas IRP2-/- mice misregulate the expression of target proteins in all tissues. Surprisingly, the RNA-binding activity of IRP1 does not increase in animals on a low-iron diet that is sufficient to activate IRP2. In animal tissues, most of the bifunctional IRP1 is in the form of cytosolic aconitase rather than an RNA-binding protein. Our findings indicate that the small RNA-binding fraction of IRP1, which is insensitive to cellular iron status, contributes to basal mammalian iron homeostasis, whereas IRP2 is sensitive to iron status and can compensate for the loss of IRP1 by increasing its binding activity. Thus, IRP2 dominates post-transcriptional regulation of iron metabolism in mammals.

Effect of glucagon-like peptide-2 (GLP-2) on diurnal SGLT1 expression.

Glucagon-like peptide 2 (GLP-2) is a 33-amino acid gut peptide that leads to villus hyperplasia and altered gene expression. We examined the effect of chronically administered GLP-2 on diurnal gene expression rhythms using the Na+/glucose cotransporter 1 (SGLT1) as the index. Animals were treated with [Gly2]GLP-2 (twice daily; 1microg/g body weight) or vehicle (control) for 10 days. Rats were killed at either 3 hr or 9 hr after light onset (ZT3 and ZT9, respectively), an interval during which SGLT1 expression exhibits a robust induction. SGLT1 mRNA expression was assessed by Northern blotting and in situ hybridization. SGLT1 protein was examined by immunofluorescence and Western blotting. Tissues from GLP-2-treated rats had increased villus height, crypt depth, and proliferation index (P < 0.05). GLP-2 administration did not alter the diurnal increase in mRNA levels of SGLT1, GLUT2, or GLUT5. However, in GLP-2-treated rats, the SGLT1 protein amount increased at both ZT3 and ZT9. Moreover, SGLT1 was preferentially localized to the apical membranes in this group. GLP-2 does not adversely affect the diurnal expression rhythm of SGLT1 and appears to increase membrane expression of the protein. These biological actions of GLP-2 may contribute to its therapeutic value in intestinal diseases.

Effect of middle cerebral artery occlusion on mRNA expression for the sodium-coupled vitamin C transporter SVCT2 in rat brain.

The sodium-vitamin C co-transporter SVCT2 is primarily responsible for the accumulation of the important antioxidant ascorbate into brain cells. In vitro studies have demonstrated strong expression of this transporter in cultured astrocytes, whereas in situ hybridization analysis has so far detected SVCT2 only in neurons. In the present study, we examined the response of SVCT2 mRNA expression in the brain to focal ischemia induced for 2 h by unilateral middle cerebral artery occlusion. The mRNA expression patterns of SVCT2 and the glutamate-activated immediate early gene Arc were investigated at 2 and 22 h after ischemia. SVCT2 and Arc mRNA expression was lost in the ischemic core at both time points. In areas outside the core, Arc was strongly up-regulated, primarily at 2 h, whereas SVCT2 showed an increase at 2 and 22 h. SVCT2 expression was increased in neurons as well as in astrocytes, providing the first evidence for SVCT2 expression in astrocytes in situ. These findings underscore the importance of ascorbate as a neuroprotective agent and may have implications for therapeutic strategies. In addition, the increase of SVCT2 in astrocytes after ischemia suggests that cultured astrocytes are exposed to chronic oxidative stress.

Tissue-engineered neomucosa: morphology, enterocyte dynamics, and SGLT1 expression topography.

BACKGROUND: The standard therapy for short bowel syndrome is total parenteral nutrition, which is expensive and associated with significant morbidity and mortality. New therapeutic approaches for this disorder are needed. We have applied the techniques of tissue engineering to develop a prototype neointestine. We hypothesized that anastomosis of this neointestine to the native bowel would result in regeneration of mucosal morphology and enterocyte dynamics. METHODS: Biodegradable polymers seeded with neonatal rat intestinal organoid units were implanted into the omenta of adult rats to form neointestinal cysts. Five weeks after implantation, side-to-side cyst-jejunal anastomoses were fashioned in one cohort of rats. Tissues were harvested from all rats at 5 months after implantation. Native jejunal (J) and non-anastomosed (N-N) and anastomosed (A-N) neointestinal tissues were assessed for morphology, epithelial cell proliferation (5-bromo-2-deoxyuridine immunohistochemistry), apoptotic rates (terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay), and SGLT1 in situ hybridization. RESULTS: Mucosal morphology, rates and topography of enterocyte proliferation, and transporter expression in A-N neointestine recapitulated those of native jejunum. Each of these features was rudimentary in N-N neointestine. CONCLUSIONS: These results suggest that the tissue-engineered neomucosa can develop structural and dynamic features of the normal jejunum. Anastomosis to the native intestine is an essential step for neomucosal development. Tissue engineering offers promise as a novel approach to the treatment of patients suffering from short bowel syndrome.

Intestinal expression of genes involved in iron absorption in humans.

Hereditary hemochromatosis (HHC) is one of the most frequent genetic disorders in humans. In healthy individuals, absorption of iron in the intestine is tightly regulated by cells with the highest iron demand, in particular erythroid precursors. Cloning of intestinal iron transporter proteins provided new insight into mechanisms and regulation of intestinal iron absorption. The aim of this study was to assess whether, in humans, the two transporters are regulated in an iron-dependent manner and whether this regulation is disturbed in HHC. Using quantitative PCR, we measured mRNA expression of divalent cation transporter 1 (DCT1), iron-regulated gene 1 (IREG1), and hephaestin in duodenal biopsy samples of individuals with normal iron levels, iron-deficiency anemia, or iron overload. In controls, we found inverse relationships between the DCT1 splice form containing an iron-responsive element (IRE) and blood hemoglobin, serum transferrin saturation, or ferritin. Subjects with iron-deficiency anemia showed a significant increase in expression of the spliced form, DCT1(IRE) mRNA. Similarly, in subjects homozygous for the C282Y HFE mutation, DCT1(IRE) expression levels remained high despite high serum iron saturation. Furthermore, a significantly increased IREG1 expression was observed. Hephaestin did not exhibit a similar iron-dependent regulation. Our data show that expression levels of human DCT1 mRNA, and to a lesser extent IREG1 mRNA, are regulated in an iron-dependent manner, whereas mRNA of hephaestin is not affected. The lack of appropriate downregulation of apical and basolateral iron transporters in duodenum likely leads to excessive iron absorption in persons with HHC.

Tissue and cellular localization of a novel polycystic kidney disease-like gene product, polycystin-L.

Polycystin-L (PCL), the third member of the polycystin family of proteins, functions as a Ca2+-modulated nonselective cation channel when expressed in Xenopus oocytes. Polycystin-1 and -2 are mutated in autosomal-dominant polycystic kidney disease (ADPKD), but the role of PCL in disease has not been determined. In this study, an anti-peptide polyclonal antiserum was generated against the carboxyl terminal domain of human PCL and used to determine the patterns of expression and distribution of PCL by indirect immunofluorescence in both developing and adult mice. The results show that PCL is predominantly expressed in adult mouse tissues and has a more restricted pattern of expression than either polycystin-1 or -2. In the kidney, PCL expression was first detected at E16, and levels increased into adulthood. Localization of PCL was predominantly found in the apical region of the principal cells of inner medullary collecting ducts. PCL was also found in discrete cell types of the retina, testis, liver, pancreas, heart, and spleen, but it was not detected in the lung. These data in combination with evidence of PCL channel activity are crucial for elucidating the physiologic role of this novel cation channel and may shed light on the function of inner medullary collecting ducts and polycystins. The expression pattern of PCL suggests that it is unlikely to be a candidate gene for ADPKD, but it remains a potential candidate for other as yet unmapped human cystic disorders.