Richard L. Leino

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.

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.

Reproductive and developmental toxicity and bioconcentration of perfluorooctanesulfonate in a partial life‐cycle test with the fathead minnow (Pimephales promelas)

Perfluorooctanesulfonate (PFOS) is a widespread environmental contaminant emanating from the production and/or metabolism of fluorinated chemicals with a variety of applications. The goal of this work was to assess the toxicity and bioconcentration of PFOS in the fathead minnow (Pimephales promelas). Sexually mature fish were exposed via the water for 21 d to 0 (control), 0.03, 0.1, 0.3, or 1 mg PFOS/L, and effects on reproductive capacity and endocrinology were assessed. To determine possible developmental effects, a subset of embryos from parental exposures at each test concentration were held for an additional 24 d in the same PFOS treatments. A concentration of I mg PFOS/L was lethal to adults within two weeks. The 21-d 50% effect concentration (95% confidence interval) for effects on fecundity of the fish was 0.23 (0.19-0.25) mg PFOS/L. Exposure to PFOS caused various histopathological alterations, most prominently in ovaries of adult females. Adult males exposed to 0.3 mg PFOS/L for 21 d exhibited decreased aromatase activity and elevated concentrations of plasma 11-ketotestosterone and testosterone. No significant adverse effects on survival or growth were observed in developing fathead minnows held for 24 d at PFOS concentrations up to 0.3 mg/L. Adult fathead minnows readily accumulated PFOS from the water. The largest concentrations of PFOS were in blood, followed by liver and then gonad; for all tissues, females accumulated higher concentrations than males. Water and tissue concentrations of PFOS associated with effects in this study exceeded those reported for samples collected from the field by two to three orders of magnitude, suggesting that the current risk of PFOS on aspects of fish reproduction and development assessed in this study would be small.

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. GLIA 22:272–281, 1998.

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. J. Neurosci. Res. 49:617–626, 1997.

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.

Ultrastructure of immature, developing, and secretory rodlet cells in fish

SummaryRodlet cells from many species of fresh water fish were examined with the electron microscope. Observations of these cells in the epithelia of intestine, gill, and other organs favor the interpretation that rodlet cells are secretory cells rather than protozoan parasites (Rhabdospora thelohani). Evidence is presented that rodlet cells develop from undifferentiated cells near the epithelial basement membranes and migrate“upward” to secrete their contents in a holocrine manner at the epithelial surface. The major secretory product is packaged in unique club-shaped sacs which contain a central core of highly electron dense material with less dense material surrounding it. Secretion often appears to be accomplished by active contraction of a 0.5 μm fibrillar border resembling smooth muscle which lies just inside of the plasma membrane. Some rodlet cells, however, merely break apart and release their contents.

Morphological and morphometrical changes in chloride cells of the gills of Pimephales promelas after chronic exposure to acid water.

SummaryFathead minnows, Pimephales promelas, were exposed for 129 days to Lake Superior water acidified with sulfuric acid by means of a flow-through toxicant injection system. The effects of chronic acid stress (pH 6.5, 6.0, 5.5, 5.0) on gill histology were examined. Most of the histological effects were seen at pH 5.5 and 5.0 and were confined primarily to changes in numbers, distribution, and morphology of chloride cells. At low pH levels there tend to be more chloride cells in the gill epithelium and an increased percentage of these cells in the secondary lamellae. In contrast to normal chloride cells, chloride cells from fish exposed to low pH frequently had apical pits while some had bulbous apical evaginations. The occurrence of structural changes in chloride cells during exposure to acid water suggests that chloride cells may be involved in acclimation to acid stress.

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.

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 when brain glucose metabolism is elevated.