Biomedical subjects

L R Drewes

Publications and source records attributed to L R Drewes.

At least 19 recordsLinked to original sources

PubMed2001

Factor(s) released by glucose-deprived astrocytes enhance glucose transporter expression and activity in rat brain endothelial cells.

Glucose transporter (GLUT) expression and regulation were studied in rat brain endothelial cells in primary culture (RBEC) and in immortalised RBE4 cells. Immunoblotting analysis showed a low expression of the endothelium-specific GLUT1 in RBEC and RBE4 cells compared to isolated brain capillaries. RBEC and RBE4 cells also expressed the GLUT3 isoform, whereas it was not present in isolated brain capillaries. No change in GLUT expression was observed in endothelial cells treated with astrocyte-conditioned medium. However, treatment with conditioned medium obtained from glucose-deprived astrocytes increased endothelial GLUT1 expression and glucose uptake. These results suggest that astrocytes submitted to hypoglycaemic conditions may release factor(s) that increase glucose uptake through the blood-brain barrier.

Animals↗

PubMed2001

Expression of monocarboxylate transporter MCT1 in normal and neoplastic human CNS tissues.

Expression of monocarboxylate transporter MCT1 was studied in archival tissues from human CNS using antibodies to the carboxyl-terminal end of MCT1. Sections of neocortex, hippocampus and cerebellum of brains from 10 adult autopsy patients who died from other than CNS disease, and from archival surgical biopsy specimens of 83 primary CNS and eight non-CNS tumors were studied. MCT1 immunoreactivity was present in microvessels and, ependymocytes of normal CNS tissues similar to that reported for MCT1 expression in rat brains. MCT1 immunoreactivity was strongest in ependymomas, hemangioblastomas and high grade glial neoplasms, and weakest in low grade gliomas. Increased MCT1 expression in high grade glial neoplasms may provide a potential therapeutic target for treatment of some CNS neoplasms.

Antibodies↗

PubMed2001

Diet-induced ketosis increases monocarboxylate transporter (MCT1) levels in rat brain.

Monocarboxylate transporter (MCT1) levels in brains of adult Long-Evans rats on a high-fat (ketogenic) diet were investigated using light and electron microscopic immunocytochemical methods. Rats given the ketogenic diet (91% fat and 9% protein) for up to 6 weeks had increased levels of the monocarboxylate transporter MCT1 (and of the glucose transporter GLUT1) in brain endothelial cells and neuropil compared to rats on a standard diet. In ketonemic rats, electron microscopic immunogold methods revealed an 8-fold greater MCT1 labeling in the brain endothelial cells at 4 weeks. Abluminal endothelial membranes were twice as heavily labeled as luminal membranes. In controls, luminal and abluminal labeling was not significantly different. The endothelial cytoplasmic compartment was sparsely labeled (<8% of total endothelial labeling) in all brains. Neuropil MCT1 staining was more intense throughout the brain in ketonemic rats, especially in neuropil of the molecular layer of the cerebellum, as revealed by avidin-biotin immunocytochemistry. This study demonstrates that adult rats retain the capacity to upregulate brain MCT1 levels. Furthermore, their brains react to a diet that increases monocarboxylate levels in the blood by enhancing their capability to take up both monocarboxylates (MCT1 upregulation) and glucose (GLUT1 upregulation). This may have important implications for delivery of fuel to the brain under stressful and pathological conditions, such as epilepsy and GLUT1 deficiency syndrome.

Animals↗

PubMed2000

Expression of large amino acid transporter LAT1 in rat brain endothelium.

The expression of the large amino acid transporter, LAT1, was investigated in brain of adult Long-Evans rats. The LAT1 transcript was readily detected in brain microvessels and choroid plexus by reverse transcription polymerase chain reaction analysis using three different gene specific primer pairs. A polyclonal affinity purified antibody against the N-terminus of LAT1 was generated in chickens and used in immunoblot and immunocytochemical analyses of brain tissue sections of adult rats. On immunoblots, the antibody detected a peptide-inhibitable 45 kDa band in a rat brain microvessel membrane preparation. It also identified the same protein band in membrane preparations of different brain structures, as well as in heart and testis, whereas the protein was absent or only faintly detectable in muscle, kidney, and liver. In brain sections, the antibody intensely labeled the luminal and abluminal membranes of brain microvessel endothelial cells in all brain areas examined including cerebral cortex, cerebellum, hippocampus, and in gray and white matter regions. These results suggest that LAT1 is involved in transcellular transport and may play an important role in large, neutral amino acid transfer across the blood-brain barrier.

Age Factors↗

PubMed1999

Monocarboxylate transporter (MCT1) abundance in brains of suckling and adult rats: a quantitative electron microscopic immunogold study.

Transcellular transport of energy substrates across the vascular endothelial cells of the brain is accomplished by integral membrane carrier proteins, such as the glucose transporter GLUT1 and the monocarboxylic acid transporter MCT1. The abundance of these proteins may vary depending on age and nutritional status. In this study we compared the expression of MCT1 in cerebral cortex of suckling and adult rats to determine whether the former, which use considerably more monocarboxylates such as lactate and ketone bodies as fuel than do older rats, correspondingly express more MCT1 than adults. Using electron microscopic immunogold methods, we found that 17-day old suckling rat pups had 25 times more MCT1 labeling in the membranes of capillary endothelial cells than adults. This transporter was nearly equally distributed in luminal and abluminal membranes with less than 10% of the immunogold particles in the endothelial cytoplasmic compartment. The suckling rats also had 15 times more immunogold particles associated with pericyte membranes and 19 times heavier labeling of membranes associated with astrocytic end feet adjacent to microvessels. Neuropil and choroid plexus were lightly labeled. Some MCT1-positive astrocyte and neuron cell bodies were observed, suggesting active synthesis of MCT1 by these cells. The potential for regulation of expression of MCTs by dietary or other factors may have important consequences for the progression and treatment of cerebrovascular disorders and other diseases.

Age Factors↗

PubMed1999

What is the blood-brain barrier? A molecular perspective. Cerebral vascular biology.

The term "blood-brain barrier" was coined over one hundred years ago as a result of the observation that vital dyes introduced into the circulation quickly penetrated and stained nearly all organs and tissues of the mammalian body except the brain which retained its pale creamy appearance. Advances in microscopy revealed that, in contrast to other vascular beds, the brain endothelial cells lining the vascular wall are tightly linked with junctional complexes that eliminate gaps or spaces between cells and prevent any free diffusion of blood-borne substances into the brain parenchymal space. The endothelial cells, situated at the interface between blood and brain, therefore, play a critical role in performing essential biological functions including transport of micro- and macronutrients, receptor-mediated signaling, leukocyte trafficking, and osmoregulation. A number of molecular components responsible for some of these unique properties have now been identified and are being characterized under physiological and disease conditions. These include the proteins involved in formation and assembly of tight junctions; the plasma membrane-embedded proteins that are responsible for transport of brain energy substrates and nutrients (glucose, monocarboxylic acids, nucleosides, amino acids, others); the multi-drug transporter protein, p-glycoprotein, and other drug-rejecting proteins that protect the brain from foreign, potentially disruptive chemicals. These and other recent findings, taken as a whole, reveal the brain endothelium as a complex and dynamic biological system, in contrast to the simple, inert and rigid barrier initially perceived.

Animals↗

PubMed1999

Distribution of monocarboxylate transporters MCT1 and MCT2 in rat retina.

Transport of lactic acid and other monocarboxylates such as pyruvate and the ketone bodies through cellular membranes is facilitated by specific transport proteins. We used chicken polyclonal antibodies to the monocarboxylate transporters-1 and -2 to determine their cellular and subcellular distributions in rat retina, and we compared these distributions to those of the glucose transporters-1 and -3. Monocarboxylate transporter-1 was most highly expressed by the apical processes of retinal pigment epithelium that surround the outer segments of the photoreceptor cells. In contrast to glucose transporter-1, monocarboxylate transporter-1 was not detected on the basal membranes of pigment epithelium. The luminal and abluminal endothelial plasma membranes in retina also exhibited heavy labeling by antibody to monocarboxylate transporter-1. In addition, this transporter was associated with the Müller cell microvilli, the plasma membranes of the rod inner segments, and all retinal layers between the inner and external limiting membranes. Monocarboxylate transporter-2 was found to be abundantly expressed on the inner (basal) plasma membrane of Müller cells and by glial cell processes surrounding retinal microvessels. This transporter was also present in the plexiform and nuclear layers but was not detected beyond the external limiting membrane. Recent studies have shown that lactic acid transport is of particular importance at endothelial and epithelial barriers where membranes of adjoining cells are linked by tight junctions. Our results suggest that monocarboxylate transporter-1 functions to transport lactate between the retina and the blood, both at the retinal endothelium and the pigment epithelium. The location of monocarboxylate transporter-2 on glial foot processes surrounding retinal vessels suggests that this transporter is also important in blood-retinal lactate exchange. In addition, the abundance of these transporters in Müller cells and synaptic (plexiform) layers suggests that they function in lactate exchange between neurons and glia, supporting the notion that lactate plays a key role in neural metabolism.

Animals↗

PubMed1999

Overexpression of monocarboxylate transporter and lactate dehydrogenase alters insulin secretory responses to pyruvate and lactate in beta cells.

Previous investigations revealed low activities of lactate dehydrogenase (LDH) and plasma membrane monocarboxylate transporters (MCT) in the pancreatic beta cell. In this study the significance of these characteristics was explored by overexpressing type A LDH (LDH-A) and/or type 1 MCT (MCT-1) in the clonal INS-1 beta cells and isolated rat islets. Inducible overexpression of LDH-A resulted in an 87-fold increase in LDH activity in INS-1 cells. Adenovirus-mediated overexpression of MCT-1 increased lactate transport activity 3.7-fold in INS-1 cells. Although overexpression of LDH-A, and/or MCT-1 did not affect glucose-stimulated insulin secretion, LDH-A overexpression resulted in stimulation of insulin secretion even at a low lactate concentration with a concomitant increase in its oxidation in INS-1 cells regardless of MCT-1 co-overexpression. Adenovirus-mediated overexpression of MCT-1 caused an increase in pyruvate oxidation and conferred pyruvate-stimulated insulin release to isolated rat islets. Although lactate did not stimulate insulin secretion from control or MCT-1-overexpressing islets, co-overexpression of LDH-A and MCT-1 evoked lactate-stimulated insulin secretion with a concomitant increase in lactate oxidation in rat islets. These results suggest that low expression of MCT and LDH is requisite to the specificity of glucose in insulin secretion, protecting the organism from undesired hypoglycemic actions of pyruvate and lactate during exercise and other catabolic states.

Adenoviridae↗

PubMed1998

TAT1 encodes a low-affinity histidine transporter in Saccharomyces cerevisiae.

Previous studies have revealed the presence of at least two histidine uptake systems in S. cerevisiae; one with high affinity and the other with low affinity for histidine. The HIP1 gene is known to encode the high affinity permease. The purpose of this study was to identify the gene that encodes the low affinity permease. A mutant strain of S. cerevisiae that is both a histidine auxotroph and a hip1 deletion mutant is unable to grow on low histidine media. This strain was transformed with a yeast cDNA library constructed in a yeast expression vector. Transformants with increased histidine transport were selected by their ability to grow on a low histidine media. Sequencing of the inserts revealed the presence of the HIP1 gene and also the presence of the TAT1 gene. Estimated Km and Vmax values for histidine transport by each system were determined. In a hip1 tat1 double mutant, the level of histidine required for growth increased eight-fold in comparison to the hip1 single mutant. Our results suggest that the TAT1-encoded protein, previously characterized as the high-affinity tyrosine permease, also acts as the low affinity histidine permease.

ATP-Binding Cassette Transporters↗

PubMed1998

Expression of the monocarboxylate transporter MCT2 by rat brain glia.

The nucleotide sequence of the rat monocarboxylate transporter MCT2 was determined from brain-derived cDNA. A polyclonal antibody was raised in chickens against the carboxyl terminal end of the deduced amino acid sequence and affinity purified. The MCT2 antibody identified a 46-kDa band on immunoblots and labeled kidney, skeletal muscle, and stomach consistent with the reported cellular expression for this transporter. Light microscopic immunocytochemistry indicated that the MCT2 transporter was abundant in glial limiting membranes, ependymocytes, and neuropil, particularly in the lacunosum molecular layer of hippocampus and the molecular layer of cerebellum. Labeled astrocytes were commonly observed in white matter. The distribution of this transporter differed in several respects from that previously reported for MCT1. MCT2 was abundantly distributed in astrocyte foot processes and was usually not detected in other cells of the cerebrovasculature, including vascular smooth muscle cells, pericytes, and endothelium. In addition, the granular layer of cerebellum, which showed little MCT1 labeling, exhibited MCT2 labeling of cellular processes in the neuropil surrounding the granule and Purkinje cells. The results lend support to the concept that astrocytes play a significant role in cerebral energy metabolism by transporting lactate and other monocarboxylates.

Animals↗

PubMed1997

Ultrastructural localization of GLUT 1 and GLUT 3 glucose transporters in rat brain.

Precise localization of glucose transport proteins in the brain has proved difficult, especially at the ultrastructural level. This has limited further insights into their cellular specificity, subcellular distribution, and function. In the present study, preembedding ultrastructural immunocytochemistry was used to localize the major brain glucose transporters, GLUTs 1 and 3, in vibratome sections of rat brain. Our results support the view that, besides being present in endothelial cells of central nervous system (CNS) blood vessels, GLUT 1 is present in astrocytes. GLUT 1 was detected in astrocytic end feet around blood vessels, and in astrocytic cell bodies and processes in both gray and white matter. GLUT 3, the neuronal glucose transporter, was located primarily in pre- and postsynaptic nerve endings and in small neuronal processes. This study: (1) affirms that GLUT 3 is neuron-specific, (2) shows that GLUT 1 is not normally expressed in detectable quantities by neurons, (3) suggests that glucose is readily available for synaptic energy metabolism based on the high concentration of GLUT 3 in membranes of synaptic terminals, and (4) demonstrates significant intracellular and mitochondrial localization of glucose transport proteins.

Animals↗

PubMed1997

Expression of monocarboxylate transporter MCT1 by brain endothelium and glia in adult and suckling rats.

A polyclonal affinity-purified antibody to the carboxyl-terminal end of the rat monocarboxylate transporter 1 (MCT1) was generated in chickens and used in immunocytochemical studies of brain tissue sections from adult and suckling rats. The antibody identified a 48-kDa band on immunoblots and stained tissue sections of heart, cecum, kidney, and skeletal muscle, consistent with the reported molecular mass and cellular expression for this transporter. In tissue sections from adult brains, the antibody labeled brain microvessel endothelial cells, ependymocytes, glial-limiting membranes, and neuropil. In brain sections from 3- to 14-day-old rats, microvessels were much more strongly labeled and neuropil was weakly labeled compared with sections from adults. Immunoelectron microscopy indicated that labeling was present on both luminal and abluminal endothelial cell plasma membranes. These results suggest that MCT1 may play an important role in the passage of lactate and other monocarboxylates across the blood-brain barrier and that suckling rats may be especially dependent on this transporter to supply energy substrates to the brain.

Aging↗

PubMed1996

Canine brain glucose transporter 3: gene sequence, phylogenetic comparisons and analysis of functional sites.

There has been a sparsity of various mammalian neuronal glucose transporter 3-encoding sequences(Glut3) available for the purposes of alignment studies. We report here a 2355-bp sequence of canine Glut3 that encodes a deduced protein of 496 amino acids (aa). The full-length canine aa sequence was compared to those of the human, mouse and rat glucose transporter 3 (Glut3), and found to be 88.3, 84.9 and 84.3% identical, respectively. However, while mouse and rat identical C-termini, the canine nd human C-termini share markedly little identity or similarity to one another, or to that of rat/mouse. The canine Glut3 sequence also exhibits 74.5% aa identity with a non-mammalian chicken Glut3 sequence. These differences in the C-termini of Glut3 among the species may result in kinetic or mechanistic differences in transport of glucose. Computer searches were made for conserved functional motifs, and a brief review of ten sites is provided. This review includes the determination of their locations in two transmembrane (TM) motifs that have been proposed for glucose transporters. The nucleotide (nt) sequence of the 5'-untranslated region (UTR) of canine Glut3 was aligned with the comparable human glut3 region and was shown to be 70% identical over a region of 129 nt just prior to the ATG start codons. A similar comparison of the 3'-UTR shows 74% identity over 350 nt immediately following the stop codons. An adenosine-uridine-binding factor (AUBF) region, which has been identified as a region of importance in mRNA stabilization, is conserved in the 3'-UTR of both canine and human Glut3. The conservation in the UTR suggests that Glut3 may be post-transcriptionally regulated.

Amino Acid Sequence↗

PubMed1996

Predicting blood-brain transport of drugs: a computational approach.

PURPOSE: This study was conducted to determine the efficacy of using nonempirical parameters in the estimation of blood-brain transport, inferred from central nervous system (CNS) activity, for a set of twenty-eight compounds. METHODS: A discriminant function analysis was used to construct three distinct models based on topological indices, a hydrogen-bonding parameter, and logP. RESULTS: These models correctly predict the CNS activity of twenty-seven of the twenty-eight compounds. CONCLUSIONS: Nonempirical parameters may be used effectively in the estimation the cerebrovascular penetration for known and newly designed drugs.

Blood-Brain Barrier↗

PubMed1996

Chronic seizures increase glucose transporter abundance in rat brain.

Pentylenetetrazole and kainic acid, seizure-inducing agents that are known to increase glucose utilization in brain, were used to produce chronic seizures in mature rats. To test the hypothesis that increased brain glucose utilization associated with seizures may alter glucose transporter expression, polyclonal carboxyl-terminal antisera to glucose transporters (GLUT1 and GLUT3) were employed with a quantitative immunocytochemical method and immunoblots to detect changes in the regional abundances of these proteins. GLUT3 abundances in control rats were found to be correlated with published values for regional glucose utilization in normal brain. Following treatment with kainic acid and pentylenetetrazole, both GLUT3 and GLUT1 increased in abundance in a region and isoform-specific manner. GLUT3 was maximal at eight hours, whereas GLUT1 was maximal at three days. Immunoblots indicated that most of the GLUT3 increase was accounted for by the higher molecular weight component of the GLUT3 doublet. The rapid response time for GLUT3 relative to GLUT1 may be related to the rapid increase in neuronal metabolic energy demands during seizure. These observations support the hypothesis that glucose transporters may be upregulated in brain under conditions when brain glucose metabolism is elevated.

Animals↗

PubMed1995

Localization of glucose transporter GLUT 3 in brain: comparison of rodent and dog using species-specific carboxyl-terminal antisera.

The carboxyl-terminal amino acid sequences of the canine and gerbil glucose transporter GLUT3 were determined and compared to the published rat sequence. Eleven of 16 amino acids comprising the carboxyl terminus of GLUT3 were found to be identical in rat and dog. However, the canine sequence "ATV" substitutes for the rat sequence "PGNA" at the end of the molecule. The gerbil sequence has 12 of 16 amino acids identical to the rat, including the PGNA terminus. Based on these sequences, four peptides were synthesized, and two polyclonal antisera (one to the canine sequence and one to the rat sequence) were raised to examine the distribution of GLUT3 in canine and rodent brain. Immunoblots of brain membrane preparations showed that both antisera identified peptide-inhibitable protein bands of molecular weight 45,000-50,000. Immunocytochemical studies demonstrated that binding sites for these antisera were abundantly distributed in neuropil in all brain regions. Areas rich in synapses and areas surrounding microvessels exhibited especially high reactivity. GLUT3 reactivity was similarly distributed in canine and rodent brain, except at the blood-brain barrier. GLUT3 was not detected in the blood-brain barrier in gerbil and rat but was present in many canine cerebral endothelial cells, particularly in cerebellum and brain stem. The carboxyl-terminal antisera employed in this study exhibited high degrees of species specificity, indicating that the three or four terminal amino acids of the immunizing peptides (ATV and PGNA) are important epitopes for binding the polyclonal antibodies. These antisera exhibited only minimal binding to brain tissue of non-target species, yet yielded similar staining patterns in neuropil of rodent and canine brain. This finding provides strong evidence that the observed staining patterns accurately reflect the distribution of GLUT3 in brain. In addition, the presence of vascular GLUT3 in dog brain suggests that the canine blood-brain barrier may be preferable to that of the rat as a model for studies of glucose transport relevant to human brain.

Amino Acid Sequence↗

PubMed1994

GLUT1 and GLUT3 gene expression in gerbil brain following brief ischemia: an in situ hybridization study.

GLUT1 and GLUT3 mRNAs in normal and post-ischemic gerbil brains were examined qualitatively and semi-quantitatively using in situ hybridization in conjunction with image analysis. Coronal brain sections at the level of the anterior hippocampus were prepared three hours, one day, and three days after animals were subjected to six min of ischemia. The sections were hybridized with vector- and PCR-generated RNA probes labeled with 35S. Microscopic evaluation of hybridized brain sections coated with autoradiographic emulsion indicated that GLUT1 mRNA was associated with brain microvessels, choroid plexus, and some ependymal cells. GLUT1 mRNA was not observed in neurons, except that one day following ischemia, this mRNA was induced in neurons of the dentate gyrus. GLUT3 mRNA was detected only in neurons. Image analysis of film autoradiograms revealed that both the GLUT1 and GLUT3 messages increased following ischemia but returned nearly to control levels by day three. In the CA1 region of the hippocampus the increase in GLUT3 mRNA was not statistically significant, and by day three the level had fallen significantly below the control, coinciding with the degeneration of the CA1 neurons. Our results suggest that the brain possesses mechanisms for induction and up-regulation of glucose transporter gene expression.

Animals↗

PubMed1993

Glucose transporter (GLUT1) expression by canine brain microvessel endothelial cells in culture: an immunocytochemical study.

We have found that immunocytochemical techniques can be used to monitor the qualitative expression of GLUT1 in cultured cells. Primary cultures of BMEC showed significant GLUT1 staining, and primary cultures of endothelial cells from a non-barrier source (aorta) were negative. BMEC express significant levels of GLUT1 in young primary cultures, thus affording an opportunity for short-term regulation studies of GLUT1 activity and expression. Immunocytochemistry may be an efficient method for screening potential GLUT1 induction and maintenance factors at the BBB. The immunocytochemical technique used is rapid and requires relatively few cells and may provide a useful adjunct to the more quantitative techniques of Western and Northern blotting.

Animals↗