AB3540P Sigma-AldrichAnti-Monocarboxylate Transporter 1 Antibody

Leukemia inhibitory factor (LIF), an interleukin-6 family neurocytokine, is up-regulated in response to different types of retinal stress and has neuroprotective activity through activation of the gp130 receptor/STAT3 pathway. We observed that LIF induces rapid, robust, and sustained activation of STAT3 in both the retina and retinal pigmented epithelium (RPE). Here, we tested whether LIF-induced STAT3 activation within the RPE can down-regulate RPE65, the central enzyme in the visual cycle that provides the 11-cis-retinal chromophore to photoreceptors in vivo. We generated conditional knock-out mice to specifically delete STAT3 or gp130 in RPE, retina, or both RPE and retina. After intravitreal injection of LIF, we analyzed the expression levels of visual cycle genes and proteins, isomerase activity of RPE65, levels of rhodopsin protein, and the rates of dark adaptation and rhodopsin regeneration. We found that RPE65 protein levels and isomerase activity were reduced and recovery of bleachable rhodopsin was delayed in LIF-injected eyes. In mice with functional gp130/STAT3 signaling in the retina, rhodopsin protein was also reduced by LIF. However, the LIF-induced down-regulation of RPE65 required a functional gp130/STAT3 cascade intrinsic to RPE. Our data demonstrate that a single cytokine, LIF, can simultaneously and independently affect both RPE and photoreceptors through the same signaling cascade to reduce the generation and utilization of 11-cis-retinal.

Brain glucose and ketone uptake was investigated in Fisher rats subjected to mild experimental ketonemia induced by a ketogenic diet (KD) or by 48 hours fasting (F). Two tracers were used, (11)C-acetoacetate ((11)C-AcAc) for ketones and (18)F-fluorodeoxyglucose for glucose, in a dual-tracer format for each animal. Thus, each animal was its own control, starting first on the normal diet, then undergoing 48 hours F, followed by 2 weeks on the KD. In separate rats on the same diet conditions, expression of the transporters of glucose and ketones (glucose transporter 1 (GLUT1) and monocarboxylic acid transporter (MCT1)) was measured in brain microvessel preparations. Compared to controls, uptake of (11)C-AcAc increased more than 2-fold while on the KD or after 48 hours F (P < 0.05). Similar trends were observed for (18)FDG uptake with a 1.9-2.6 times increase on the KD and F, respectively (P < 0.05). Compared to controls, MCT1 expression increased 2-fold on the KD (P < 0.05) but did not change during F. No significant difference was observed across groups for GLUT1 expression. Significant differences across the three groups were observed for plasma beta-hydroxybutyrate (beta-HB), AcAc, glucose, triglycerides, glycerol, and cholesterol (P < 0.05), but no significant differences were observed for free fatty acids, insulin, or lactate. Although the mechanism by which mild ketonemia increases brain glucose uptake remains unclear, the KD clearly increased both the blood-brain barrier expression of MCT1 and stimulated brain (11)C-AcAc uptake. The present dual-tracer positron emission tomography approach may be particularly interesting in neurodegenerative pathologies such as Alzheimer\'s disease where brain energy supply appears to decline critically.

Embryonic stem cells (ESCs) are established from the inner cell mass of preimplantation embryos, are capable of self-renewal, and exhibit pluripotency. Given these unique properties, ESCs are expected to have therapeutic potential in regenerative medicine and as a powerful tool for in vitro differentiation studies of stem cells. Various growth factors and extracellular matrix components regulate the pluripotency and differentiation of ESC progenies. Thus, the cell surface receptors that bind these regulatory factors are crucial for the precise regulation of stem cells. To identify membrane proteins that are involved in the regulation of pluripotent stem cells, the membrane proteins of murine ESCs cultured with or without leukemia inhibitory factor (LIF) were purified and analyzed by quantitative proteomics. 2-D PAGE-based analysis using fluorescently labeled proteins and shotgun-based analysis with isotope-labeled peptides identified 338 proteins, including transmembrane, membrane-binding, and extracellular proteins, which were expressed specifically in pluripotent or differentiated murine ESCs. Functions of the identified proteins revealed cell adhesion molecules, channels, and receptors, which are expected to play important roles in the maintenance of murine ESC pluripotency. Membrane proteins that are expressed in pluripotent ESCs but not in differentiated cells such as Slc16a1 and Bsg could be useful for the selection of the stem cells in vitro.

Skeletal muscles of elderly individuals show fatigue resistance and reduced lactate accumulation compared with those of young subjects during activities that recruit a small amount of muscle mass. To explore the mechanism underlying the functional changes in aged muscle, we focused on lactate metabolic properties, including monocarboxylate transporter (MCT) 1 and MCT4, in muscles from old and young control rats and hindlimb-suspended young rats. MCT1 expression was lower in soleus (SOL) of old rats than in SOL of young control rats, but was similar in young control and hindlimb-suspended rats. MCT4 expression was lower in extensor digitorum longus (EDL) of old rats than in that of young control rats, but did not differ between young control and hindlimb-suspended rats. The ratio of lactate dehydrogenase to citrate synthase activities was higher in SOL of hindlimb-suspended and old rats than in SOL of young control rats, and was lower in EDL of old rats than in those of young control and hindlimb-suspended rats. Our data suggest that aging causes metabolic changes that can reduce lactate accumulation during exercise and increase fatigue resistance in skeletal muscle, and that these changes result from aging rather than from inactivity.

We examine the influence of the cytosolic and membrane-bound contents of carbonic anhydrase (CA; CAII, CAIII, CAIV, and CAXIV) and the muscle content of proteins involved in lactate and proton transport [monocarboxylate transporter (MCT) 1, MCT4, and Na(+)/H(+) exchanger 1 (NHE1)] on work capacity during supramaximal exercise. Eight healthy, sedentary subjects performed exercises at 120% of the work rate corresponding to maximal oxygen uptake (W(max)) until exhaustion in placebo (Con) and metabolic alkalosis (Alk) conditions. The total (W(tot)) and supramaximal work performed (W(sup)) was measured. Muscle biopsies were obtained before and immediately after standardized exercises (se) at 120% W(max) in both conditions to determine the content of the targeted proteins, the decrease in muscle pH (DeltapH(m)), and the muscle lactate accumulation ([Lac](m)) per joule of W(sup) (DeltapH(m)/W(sup-se) and Delta[Lac](m)/W(sup-se), respectively) and the dynamic buffer capacity. In Con, W(sup) was positively [corrected] correlated with [corrected] MCT1, and tended to be positively correlated with MCT4 and NHE1. CAII + CAIII were correlated positively with DeltapH(m)/W(sup-se) and negatively with Delta[Lac](m)/W(sup-se), while CAIV was positively related to W(tot). The changes in W(sup) with Alk were correlated positively with those in dynamic buffer capacity and negatively with W(sup) in Con. Performance improvement with Alk was greater in subjects having a low content of proteins involved in pH regulation and lactate/proton transport. These results show the importance of pH regulating mechanisms and lactate/proton transport on work capacity and the role of the CA to delay decrease in pH(m) and accumulation in [Lac](m) during supramaximal exercise in humans.

1. The present study was designed to examine the role of calcineurin in muscle metabolic components by the administration of the specific calcineurin inhibitor cyclosporine A (CsA) to rats. 2. Male Wistar rats were divided into either a CsA-treated group (CT) or a vehicle-treated group (VT). Cyclosporine A was administered subcutaneously to rats at a rate of 25 mg/kg bodyweight per day for 10 successive days. Thereafter, changes in muscle enzyme activities and glucose transporter (GLUT)-4 and monocarboxylate transporter (MCT)-1 and MCT-4 proteins in the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles were examined. 3. There was a significant increase in MCT-1 and MCT-4 proteins in the soleus muscle in the CT group, but not in the EDL muscle. The activities of hexokinase, pyruvate kinase and lactate dehydrogenase in the soleus muscle also increased significantly in the CT group, but a similar increase in enzyme activity was not seen in EDL muscle. The activities of citrate synthase or malate dehydrogenase and the GLUT-4 protein content were not altered by CsA treatment in either the soleus or EDL muscles. 4. These results seem to imply that calcineurin negatively regulates the components of glucose/lactate metabolism, except for GLUT-4, especially in slow-twitch muscle.

PURPOSE: The amphiphilic block copolymer Pluronic P85 (P85) increases the permeability of the blood-brain barrier (BBB) with respect to a broad spectrum of drugs by inhibiting the drug efflux transporter, P-glycoprotein (Pgp). In this regard, P85 serves as a promising component for CNS drug delivery systems. To assess the possible effects of P85 on other transport systems located in the brain, we examined P85 interactions with the glucose (GLUT1) and monocarboxylate (MCT1) transporters. METHODS: Polarized monolayers of primary cultured bovine brain microvessel endothelial cells (BBMEC) were used as an in vitro model of the BBB. 3H-2-deoxy-glucose and 14C-lactate were selected as GLUT1 and MCT1 substrates, respectively. The accumulation and flux of these substrates added to the luminal side of the BBMEC monolayers were determined. RESULTS: P85 has little effect on 3H-2-deoxy-glucose transport. However, a significant decrease 14C-lactate transport across BBMEC monolayers is observed. Histology, immunohistochemistry, and enzyme histochemistry studies show no evidence of P85 toxicity in liver, kidney, and brain in mice. CONCLUSIONS: This study suggests that P85 formulations do not interfere with the transport of glucose. This is, probably, due to compensatory mechanisms in the BBB. Regarding the transport of monocarboxylates, P85 formulations might slightly affect their homeostasis in the brain, however, without any significant toxic effects.

Lactate transport across cell membranes is mediated by a family of proton-coupled monocarboxylate transporters (MCTs). The retinal pigment epithelium (RPE) expresses a unique member of this family, MCT3. A portion of the human MCT3 gene was cloned by polymerase chain reaction using primers designed from rat RPE MCT3 cDNA sequence. The human genomic sequence was used to design primers to clone human MCT3 cDNA and to identify a bacterial artificial chromosome clone containing the human MCT3 gene. The human MCT3 cDNA contained a 1512-nucleotide open reading frame with a deduced amino sequence 85% identical to rat MCT3. Comparison of the cDNA and genomic sequences revealed that the MCT3 gene was composed of five exons distributed over 5 kb of DNA. The exon-intron borders were conserved between the human and the chicken MCT3 genes. Using radiation hybrid mapping, the MCT3 gene was mapped to chromosome 22 between markers WI11639 and SGC30687. A search of chromosome 22 in the Sanger Centre database confirmed the location of the human MCT3 gene at 22q12.3-q13.2.

Although glucose is the major metabolic fuel needed for normal brain function, monocarboxylic acids, i.e., lactate, pyruvate, and ketone bodies, can also be utilized by the brain as alternative energy substrates. In most mammalian cells, these substrates are transported either into or out of the cell by a family of monocarboxylate transporters (MCTs), first cloned and sequenced in the hamster. We have recently cloned two MCT isoforms (MCT1 and MCT2) from a mouse kidney cDNA library. Northern blot analysis revealed that MCT1 mRNA is ubiquitous and can be detected in most tissues at a relatively constant level. MCT2 expression is more limited, with high levels of expression confined to testes, kidney, stomach, and liver and lower levels in lung, brain, and epididymal fat. Both MCT1 mRNA and MCT2 mRNA are detected in mouse brain using antisense riboprobes and in situ hybridization. MCT1 mRNA is found throughout the cortex, with higher levels of hybridization in hippocampus and cerebellum. MCT2 mRNA was detected in the same areas, but the pattern of expression was more specific. In addition, MCT1 mRNA, but not MCT2, is localized to the choroid plexus, ependyma, microvessels, and white matter structures such as the corpus callosum. These results suggest a differential expression of the two MCTs at the cellular level.

A cDNA clone which encodes a monocarboxylate transporter (ratMCT1) was isolated from a rat small intestinal cDNA library, which was screened by using full-length MCT1 cDNA of Chinese hamster ovary cells. The ratMCT1 cDNA was sequenced and predicted a protein of 494 amino acids with twelve potential transmembrane domains. The amino acid sequence showed 93.1% and 84.6% identity to the hamster and human monocarboxylate transporters, respectively. When expressed in Xenopus laevis oocytes, the ratMCT1 cRNA caused a significant increase in the uptake of radiolabeled lactic acid. Poly(A)+ RNA transcripts hybridizing to the ratMCT1 cDNA were detected in rat brain, heart, kidney, lung, muscle and brain capillaries. These results indicate that MCT1 contributes to pH-dependent and carrier-mediated transport of monocarboxylic acids in many tissues, not just in the small intestine.