Doaa Kirat

Monocarboxylate transporter 1 (MCT1) plays a direct role in short-chain fatty acids absorption in caprine rumen

Despite the importance of short‐chain fatty acids (SCFA) in maintaining the ruminant physiology, the mechanism of SCFA absorption is still not fully studied. The goal of this study was to elucidate the possible involvement of monocarboxylate transporter 1 (MCT1) in the mechanism of SCFA transport in the caprine rumen, and to delineate the precise cellular localization and the level of MCT1 protein along the entire caprine gastrointestinal tract. RT‐PCR revealed the presence of mRNA encoding for MCT1 in all regions of the caprine gastrointestinal tract. Quantitative Western blot analysis showed that the level of MCT1 protein was in the order of rumen ≥ reticulum > omasum > caecum > proximal colon > distal colon > abomasum > small intestine. Immunohistochemistry and immunofluorescence confocal analyses revealed widespread immunoreactive positivities for MCT1 in the caprine stomach and large intestine. Amongst the stratified squamous epithelial cells of the forestomach, MCT1 was predominantly expressed on the cell boundaries of the stratum basale and stratum spinosum. Double‐immunofluorescence confocal laser‐scanning microscopy confirmed the co‐localization of MCT1 with its ancillary protein, CD147 in the caprine gastrointestinal tract. In vivo and in vitro functional studies, under the influence of the MCT1 inhibitors, p‐chloromercuribenzoate (pCMB) and p‐chloromercuribenzoic acid (pCMBA), demonstrated significant inhibitory effect on acetate and propionate transport in the rumen. This study provides evidence, for the first time in ruminants, that MCT1 has a direct role in the transepithelial transport and efflux of the SCFA across the stratum spinosum and stratum basale of the forestomach toward the blood side.

Dietary pectin up-regulates monocaboxylate transporter 1 in the rat gastrointestinal tract

This work was undertaken to study the effect of pectin feeding on the expression level, cellular localization and functional activity of monocarboxylate transporter 1 (MCT1) in the gastrointestinal tract of rats. The results indicated that MCT1 protein level was significantly increased along the entire length of the gastrointestinal tract of pectin‐fed rats in comparison with control animals. Immunohistochemical analysis revealed an increase in MCT1 in the stratified squamous epithelia of the forestomach as well as in the basolateral membranes of the cells lining the gastric pit of the glandular stomach of pectin‐fed rats when compared with control animals. The parietal cells, which showed barely any or no detectable MCT1 in the control group, exhibited a strong intensity of MCT1 on the basolateral membranes in pectin‐fed rats. In the small intestine of pectin‐fed rats, strong immunopositivity for MCT1 was detected in the brush border and basolateral membranes of the absorptive enterocytes lining the entire villi, while in control rats, weak reactivity was detected on the brush border membrane in a few absorptive enterocytes in the villus tip. In the large intestine of control animals, MCT1 was detected on the basolateral membranes of the epithelia lining the caecum and colon. This staining intensity was markedly increased in pectin‐fed rats, along with the appearance of strong reactivity for MCT1 on the apical membranes of the surface and crypt epithelia of caecum and colon. Our results also showed that MCT1 co‐localizes with its chaperone, basigin (CD147), in the rat gastrointestinal tract, and that the pectin feeding increased the expression of CD147. In vivo functional studies revealed an enhanced acetate absorption in the colon of pectin‐fed in comparison with control animals. We conclude that MCT1 is up‐regulated along the gastrointestinal tract of pectin‐fed rats, which might represent an adaptive response to the increased availability of its substrates.

Monocarboxylate transporter 1 (MCT1) mediates transport of short‐chain fatty acids in bovine caecum

The present study was undertaken to investigate the functional role of monocarboxylate transporter 1 (MCT1) in the ruminant large intestine. Messenger RNA encoding for MCT1 was verified by reverse transcriptase‐polymerase chain reaction in caecum, proximal colon and distal colon of adult cattle. Both immunohistochemistry and confocal laser microscopy verified that the MCT1 protein was abundant in the surface epithelium of the large intestine, and the amount decreased from the opening of the crypt to its base. In the immunopositive cells, MCT1 was primarily localized in the basolateral membranes of epithelium lining the large intestine. Western blotting indicated that the levels of MCT1 protein were highest in the caecum, followed by proximal colon and then distal colon. In vitro studies were conducted to elucidate the possible involvement of MCT1 in the transport of short‐chain fatty acids (SCFA) across the isolated mucosal sheets of cattle caecum using the Ussing chamber technique. Acetate absorption was found to be pH dependent, and the rate of acetate absorption increased as pH decreased. The serosal application of the MCT1 inhibitor ‘p‐chloromercuribenzoic acid (pCMB)’ significantly reduced the transport of acetate across the caecal epithelium of cows. In addition, the transport of acetate was significantly reduced in the presence of its analogue, propionate, indicating that acetate and propionate compete for binding to the same transporter. The results show that MCT1 is a major route for SCFA efflux across the basolateral membrane of bovine large intestine and that it could play a role in the regulation of intracellular pH.

Presence of ten isoforms of monocarboxylate transporter (MCT) family in the bovine adrenal gland

This study provides novel information regarding the existence and precise cellular localization of various monocarboxylate transporters (MCTs) in the mammalian adrenal gland. RT-PCR results revealed that 10 MCT isoforms, namely MCT1, MCT2, MCT3, MCT4, MCT5, MCT8, MCT9, MCT10, MCT13, and MCT14 are expressed in the bovine adrenal gland. MCTs (MCT1-MCT8) proteins were examined by Western blot analysis in the bovine adrenal gland. The precise cellular localization of six MCT isoforms (MCT1-MCT5 and MCT8) within the different zones of the adrenal gland has been determined by immunohistochemical and immunofluorescence confocal laser microscopy analyses. To gain insight on the species differences for the expression profiles of MCT isoforms in this vital organ, we also examined the expression and cellular localization of MCT1-MCT8 in the rat adrenal gland. Some discrepancies in MCTs profiles between cattle and rat have been observed in the different zones of the adrenal gland. The tissue distribution pattern of MCT isoforms in the steroid-secreting adrenal cortex and catecholamine-secreting adrenal medulla suggests that they may play distinct roles in the regulation of the different hormone biosynthesis in the adrenal gland. Also, it is possible that different MCT isoforms in adrenal gland can be differentially regulated under acute or chronic conditions. This report can form the basis for future research on the regulation of these transporters in the adrenal gland.

Expression of Monocarboxylate Transporter 1 in Oral and Ocular Canine Melanocytic Tumors

Solid tumors are composed of a heterogeneous population of cells surviving in various concentrations of oxygen. In a hypoxic environment, tumor cells generally up-regulate glycolysis and, therefore, generate more lactate that must be expelled from the cell through proton transporters to prevent intracellular acidosis. Monocarboxylate transporter 1 (MCT1) is a major proton transporter in mammalian cells that transports monocarboxylates, such as lactate and pyruvate, together with a proton across the plasma membrane. Melanocytic neoplasia occurs frequently in dogs, but the prognosis is highly site-dependent. In this study, 50 oral canine melanomas, which were subdivided into 3 histologic subtypes, and 17 ocular canine melanocytic neoplasms (14 melanocytomas and 3 melanomas) were used to examine and compare MCT1 expression. Immunohistochemistry using a polyclonal chicken anti-rat MCT1 antibody showed that most oral melanoma exhibited cell membrane staining, although there were no significant differences observed among the 3 histologic subtypes. In contrast, the majority of ocular melanocytic tumors were not immunoreactive. Additionally, we documented the presence of a 45-kDa band in cell membrane protein Western blots, and sequencing of a reverse transcriptase polymerase chain reaction band of expected size confirmed its identity as a partial canine MCT1 transcript in 3 oral tumors. Increased MCT1 expression in oral melanomas compared with ocular melanocytic tumors may reflect the very different biology between these tumors in dogs. These results are the first to document canine MCT1 expression in canine tumors and suggest that increased MCT1 expression may provide a potential therapeutic target for oral melanoma.

Monocarboxylate transporter genes in the mammary gland of lactating cows

This study is the first to examine the expression of the 14 monocarboxylate transporter genes (MCT1–MCT14) in the mammary gland of mammals. RT-PCR, Western blot, immunohistochemistry, and immunofluorescence confocal laser microscopy were applied in a comprehensive approach to assess the expression and cellular localization of MCTs in the mammary gland of lactating cattle. RT-PCR revealed the existence of nine MCT isoforms, namely MCT1, MCT2, MCT3, MCT4, MCT5, MCT8, MCT10, MCT13, and MCT14 in cow mammary gland. The amplified cDNA segments were confirmed by sequence analysis and deposited in the GenBank. Using the commercially available antibodies against MCT1–MCT8, Western blotting verified the protein expression of MCT1, MCT2, MCT3, MCT4, MCT5, and MCT8 in the cow mammary gland. The precise cellular localization of the identified MCT proteins showed that both MCT1 and MCT2 were basolaterally localized on the cow mammary alveolar epithelial cells. In contrast, MCT4 protein signal was expressed on the apical membrane of these alveolar epithelia. MCT8, however, was predominantly localized on the basolateral membranes of the lactocytes, along with its weak labeling on the apical membrane of the same cells. No immunoreactive staining for MCT3 and MCT5 proteins could be detected histochemically in lactating bovine mammary tissue. Additionally, we proved the colocalization of CD147 with both MCT1 and MCT4 on the boundaries of the cow mammary alveolar epithelia. The existence and localization pattern of MCT genes in the mammary gland of lactating cows suggest their possible involvement in the transport of essential elements required for milk synthesis and secretion.

Expression and cellular localization of monocarboxylate transporters (MCT2, MCT7, and MCT8) along the cattle gastrointestinal tract

Fourteen members of the monocarboxylate transporter (MCT, SLC16) family have been identified, each having a different tissue distribution and substrate specificity. The expression of monocarboxylate transporters MCT1 and MCT4 have been studied in the gastrointestinal tract of ruminants; however, details of the expression of other MCT isoforms in the various parts of ruminant gastrointestinal tract are lacking. Reverse transcription with the polymerase chain reaction was used to study the regional distribution of MCT2, MCT3, and MCT5-MCT14 in the cattle gastrointestinal tract and verified the existence of MCT mRNA transcripts for MCT2, MCT3, MCT4, MCT7, MCT8, MCT9, MCT10, MCT13, and MCT14 in the ruminal and abomasal epithelia, mRNA transcripts for MCT2, MCT3, MCT4, MCT7, MCT8, MCT10, MCT13, and MCT14 in the jejunum, and mRNA transcripts for MCT2, MCT3, MCT4, MCT7, MCT8, MCT13, and MCT14 in the caecum of cattle. At the cellular level, immunohistochemical studies localized MCT2, MCT7, and MCT8 proteins in the cattle rumen, abomasum, jejunum, and caecum. This is the first study to detect the expression of various MCT isoforms in the gastrointestinal tract of a ruminant species. Our data suggest that these transporter proteins are involved in essential physiologic processes and are possible molecular targets for studying the regulation of the transport of short-chain monocarboxylates, aromatic amino acids, and thyroid hormones across the gastrointestinal tract of cattle.

Effect of pectin feeding on monocarboxylate transporters in rat adrenal gland

We have recently proved the expression and localization of seven monocarboxylate transporters (MCT1, MCT2, MCT3, MCT4, MCT5, MCT7, and MCT8) in the rat adrenal gland. So far, there are no data reporting possible regulation of any MCT isoform in the adrenal gland. Pectin is a soluble dietary fiber that is known to exert a hypocholesterolemic effect and increases the short chain fatty acids production in the large intestine. This work aimed to study the effect of pectin feeding on the expression of MCTs (MCT1–MCT5, MCT7, and MCT8) and their cellular distribution in rat adrenal gland. Western blotting demonstrated significant increase in the expression levels of MCT1, MCT2, MCT4, MCT5, and MCT7 in pectin-fed rats in comparison with the controls. Immunohistochemistry revealed extended distribution and distinctive increase in the immunoreactivities of MCT1, MCT2, MCT4, MCT5, and MCT7 in the adrenal cortical zones, besides the increase in the immunoreactive intensity of MCT5 and MCT7 in the adrenal medulla of pectin-fed versus control rats. Interestingly, zona glomerulosa which did not show any reactivity for MCT1 or MCT2 in controls, exhibited marked immunopositivities for both MCT1 and MCT2 in pectin-fed rats. MCT3 and MCT8, however, did not show significant changes in their expression levels between pectin-fed and control rats. Our data is the first to describe the up regulation of various MCTs in rat adrenal gland under the influence of pectin feeding. This up regulation might be a compensatory response to the hypocholesterolemic effect of pectin in order to maximize the intracellular availability of acetate. This article suggests that monocarboxylate transporters have an important physiological role in the regulation of adrenal hormones as well as in cholesterol homeostasis.

The monocarboxylate transporters exist in the cattle endocrine pancreas

Extensive studies are published concerning the distribution of monocarboxylate transporters (MCTs) in various animal issues including ruminants; nonetheless, nothing is known about their cellular expression and localization in the ruminant pancreas. The present study was carried out to examine the expression and cellular localization of all the fourteen MCT isoforms in cattle pancreas. RT-PCR verified the existence of mRNA transcripts for eight MCT isoforms, namely, MCT1, MCT2, MCT3, MCT4, MCT5, MCT8, MCT13, and MCT14 in cattle pancreas. Western blotting analysis confirmed the protein expression of these eight MCTs in the cattle pancreatic tissue. Immunohistochemical study, within the whole pancreas, was conducted to localize the eight MCTs identified, and the results showed strong positive immunoreactive staining for MCT1, MCT2, MCT4, MCT5, MCT13, and MCT14 on nearly all the islet cells of Langerhans, while we could not detect immunopositive signals in the acinar cells with any of MCTs antibodies used. This study, for the first time, showed the cellular localization and expression of MCT1–MCT5, MCT8, MCT13, and MCT14 within the ruminant pancreas. The distribution and expression pattern of MCT1, MCT2, MCT4, and CD147 in the cattle pancreas are different from that previously published on monogastric pancreas. Our study suggested that MCT1, MCT2, MCT4, MCT5, MCT13, and MCT14 may participate in the regulation of the pancreatic endocrine secretions in ruminants.

Monocarboxylate transporter 1 (MCT1)in calf and sheep stomach

This study focused on detection of monocarboxylate transporter 1 (MCT1) in mucosal tissues from the four compartments of stomach in calves and sheep on both protein and mRNA levels using Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. MCT1 was more expressed in the forestomach (rumen, reticulum, omasum) than in true stomach (abomasum) of both animals, and it was considerably more abundant in abomasum of adult sheep than of calves. The results suggest that MCT1 can be regulated by dietary changes and its physiological function is postulated to infl uence the transport of SCFA and their metabolites in ruminant stomach.