GLUT7: a new intestinal facilitated hexose transporter

The very last member of the SLC2A gene family of facilitated hexose transporters to be cloned was SLC2A7 (hGLUT7). It has been assigned to the class II of the GLUT family on the basis of sequence similarity, and its closest family member is GLUT5, an intestinal fructose transporter. GLUT7 is primarily expressed in the small intestine and colon, although mRNA has been detected in the testis and prostate as well. The protein is expressed in the apical membrane of the small intestine and colon, and it has a high affinity (<0.5 mM) for glucose and fructose. The abundance of the protein in the small intestine does change in parallel with the dietary carbohydrate. However, the distribution of GLUT7 along the small intestine does not entirely match with the availability of glucose and fructose, suggesting that the physiological substrate for this transporter has yet to be identified. Unlike GLUT13, the proton-coupled myoinositol transporter (HMIT), there is no evidence for the coupling of protons to the hexose movement via GLUT7. One area of study in which GLUT7 has provided a useful comparison with GLUT1 has been in the development of the hypothesis that the facilitated hexose transporters may have a selectivity filter at the exofacial opening of the translocation pore, which helps to determine which hexoses can be transported. If substantiated, the elucidation of this mechanism may prove useful in the design of hexose analogs for use in cancer imaging and therapeutics.     

Comparative expression of hexose transporters (SGLT1, GLUT1, GLUT2 and GLUT5) throughout the mouse gastrointestinal tract

Hexose transporters play a pivotal role in the absorption of food-derived monosaccharides in the gastrointestinal tract. Although a basic knowledge of the hexose transporters has already been gained, their detailed distribution and comparative intensities of expression throughout the gastrointestinal tract have not been fully elucidated. In this study, we quantitatively evaluated the expression of SGLT1, GLUT1, GLUT2, and GLUT5 by in situ hybridization and real-time PCR techniques using a total of 28 segments from the gastrointestinal tract of 9-week-old mice. GLUT2 and GLUT5 mRNA expressions were detected predominantly from the proximal to middle parts of the small intestine, showing identical expression profiles, while SGLT1 mRNA was expressed not only in the small intestine but also in the large intestine. Notably, GLUT1 mRNA was expressed at a considerable level in both the stomach and large intestine but was negligible in the small intestine. Immunohistochemistry demonstrated the polarized localization of hexose transporters in the large intestine: SGLT1 on the luminal surface and GLUT1 on the basal side of epithelial cells. The present study provided more elaborate information concerning the localization of hexose transporters in the small intestine. Furthermore, this study revealed the significant expression of glucose transporters in the large intestine, suggesting the existence of the physiological uptake of glucose in that location in mice.     

Expression of mRNA for glucose transport proteins in jejunum, liver, kidney and skeletal muscle of pigs

Although pigs are adapted to starch-rich diets and have high turnover rates of glucose, very scarce information is available on the molecular basis of glucose transport. Therefore, the present study attempted a systematic screening for the presence of mRNA of glucose transport proteins in main organs of glucose absorption, production and conservation. From the members of the solute carrier family SLC5A (sodium glucose cotransporter), the porcine jejunum was positive for SGLT1 and SGLT3, but also contained detectable levels of SGLT5. Liver contained SGLT1, SGLT5, traces of SGLT3 and, in one of five pigs, SGLT2. Kidney contained SGLT1, SGLT2, SGLT3, SGLT5 and hardly detectable levels of SGLT4. Skeletal muscle showed weak signals for SGLT3 and SGLT5. Screening for members of the SLC2A family (facilitated glucose transporter) in intestine revealed the presence of mRNA for GLUT1, GLUT2, GLUT5, GLUT7 and GLUT8, while GLUT3, GLUT4, GLUT10 and GLUT11 were also detectable. The liver contained GLUT1, GLUT2 and GLUT8 mRNA, while GLUT3, GLUT4, GLUT5, GLUT10 and GLUT11 were poorly detectable. The kidney was positive for GLUT1, GLUT2, GLUT5, GLUT8 and GLUT11, but traces of GLUT3, GLUT4 and GLUT10 could also be detected. Skeletal muscle had the strongest signal for GLUT4, while GLUT1, GLUT3, GLUT5, GLUT8, GLUT10 and GLUT11 showed weak signals. A total of 12 unique partial cDNA sequences were submitted to GenBank. In conclusion, this study provides molecular insight into the organ-specific expression of glucose transporters in pigs and thus sheds light on the way of glucose handling in this omnivorous species.     

GLUT14, a duplicon of GLUT3, is specifically expressed in testis as alternative splice forms

We have identified and cloned GLUT14, a novel member of the glucose transporter family. GLUT14 (SLC2A14) maps to chromosome 12p13.3 (17.1M), about 10 Mb upstream of GLUT3, with which it shares remarkable identity. Until now GLUT14 was thought to be a pseudogene. It consists of 11 exons with a genomic organization similar to that of GLUT3 and likely resulted from a duplication of GLUT3. GLUT14 has two alternatively spliced forms; the shorter form of GLUT14 (GLUT14-S) consists of 10 exons and produces a 497-amino-acid protein that is 94.5% identical to GLUT3. The long form (GLUT14-L) has an additional exon and codes for a protein with 520 amino acids that differs from GLUT14-S only at the N-terminus. GLUT14-S/L contain 12 putative membrane-spanning helices along with sugar-transporter signature motifs that have previously been shown to be essential for sugar transport activity. The putative glycosylation sites of GLUT14-S/L are present in loop 1. In contrast to the expression of GLUT3 in many tissues, both isoforms of GLUT14 are specifically expressed in testis. The mRNA level of GLUT14 in testis is about four times higher than that of GLUT3. Interestingly, the ortholog of GLUT14 is not found in mice. The multiple duplications of GLUT genes suggest that the GLUT family probably emerged by gene duplications and mutations during evolution in different lineages.     

The glucose transport facilitator GLUT8 is predominantly associated with the acrosomal region of mature spermatozoa

The glucose transporter 8 (GLUT8) is a recently identified member of the family of sugar transport facilitators. In human tissues GLUT8 is predominantly expressed in testis in a gonadotropin-dependent manner. It is shown here that the onset of mRNA synthesis of GLUT8 during the maturation of mouse testis coincides with the appearance of mature spermatozoa. Furthermore, immunohistochemistry with antiserum against the C-terminus of GLUT8 indicated that the protein was associated with spermatozoa within the seminiferous and the epididymal tubules. The GLUT8 immunoreactivity was detected within the head of mouse and human spermatozoa in the acrosomal region, and appeared to be located at the plasma membrane as well as within the cells. This specific expression and localization of GLUT8 suggests that the transport facilitator plays a major role in the fuel supply of mature spermatozoa, and that it is a potential target for inhibition of sperm cell function.     

Two human ACAT2 mRNA variants produced by alternative splicing and coding for novel isoenzymes

Acyl coenzyme A：cholesterol acyltransferase 2 （ACAT2） plays an important role in cholesterol absorption. Human ACAT2 is highly expressed in small intestine and fetal liver, but its expression is greatly diminished in adult liver. The full-length human ACAT2 mRNA encodes a protein, designated ACAT2a, with 522 amino acids. We have previously reported the organization of the human ACAT2 gene and the differentiation-dependent promoter activity in intestinal Caco-2 cells. In the current work, two human ACAT2 mRNA variants produced by alternative splicing are cloned and predicted to encode two novel ACAT2 isoforms, named ACAT2b and ACAT2c, with 502 and 379 amino acids, respectively. These mRNA variants differ from ACAT2a mRNA by lack of the exon 4 （ACAT2b mRNA） and exons 4-5 plus 8-9-10 （ACAT2c mRNA）. Significantly, comparable amounts of the alternatively spliced ACAT2 mRNA variants were detected by RT- PCR, and Western blot analysis confirmed the presence of their corresponding proteins in human liver and intestine cells. Furthermore, phosphorylation and enzymatic activity analyses demonstrated that the novel isoenzymes ACAT2b and ACAT2c lacked the phosphorylatable site SLLD, and their enzymatic activities reduced to 25%-35% of that of ACAT2a. These evidences indicate that alternative splicing produces two human ACAT2 mRNA variants that encode the novel ACAT2 isoenzymes. Our findings might help to understand the regulation of the ACAT2 gene expression under certain physiological and pathological conditions.     

Cloning and functional characterization of the human GLUT7 isoform SLC2A7 from the small intestine

Facilitated glucose transporters (GLUTs) mediate transport of sugars across cell membranes by using the chemical gradient of sugars as the driving force. Improved cloning techniques and database analyses have expanded this family of proteins to a total of 14 putative members. In this work a novel hexose transporter isoform, GLUT7, has been cloned from a human intestinal cDNA library by using a PCR-based strategy (GenBank accession no. AY571960). The encoded protein is comprised of 524 amino acid residues and shares 68% similarity and 53% identity with GLUT5, its most closely related isoform. When GLUT7 was expressed in Xenopus oocytes, it showed high-affinity transport for glucose (K(m) = 0.3 mM) and fructose (IC(50) = 0.060 mM). Galactose, 2-deoxy-d-glucose, and xylose were not transported. Uptake of 100 microM d-glucose was not inhibited by 200 microM phloretin or 100 microM cytochalasin B. Northern blotting indicated that the mRNA for GLUT7 is present in the human small intestine, colon, testis, and prostate. Western blotting and immunohistochemistry of rat tissues with an antibody raised against the predicted COOH-terminal sequence confirmed expression of the protein in the small intestine and indicated that the transporter is predominantly expressed in the enterocytes' brush-border membrane. The unusual substrate specificity and close sequence identity with GLUT5 suggest that GLUT7 represents an intermediate between class II GLUTs and the class I member GLUT2. Comparison between these proteins may provide key information as to the structural determinants for the recognition of fructose as a substrate.     

Differential regulation of glucose transporter gene expression in adipose tissue or septic rats.

To understand the molecular mechanisms responsible for the sepsis-induced enhanced glucose uptake, we have examined the levels of GLUT4 and GLUT1 mRNA and protein in the adipose tissue of septic animals. Rats were challenged with a nonlethal septic insult where euglycemia was maintained and hexose uptake in adipose tissue was markedly elevated. Northern blot analysis of total RNA isolated from epididymal fat pads indicated differential regulation of the mRNA content for the two transporters: GLUT1 mRNA was increased 2.6 to 4.6-fold, while GLUT4 mRNA was decreased by 2.5 to 2.9-fold. Despite the difference in mRNA levels, both GLUT1 and GLUT4 protein were down regulated in plasma membranes (40% and 25%, respectively) and microsomal membranes (42% and 25%, respectively) of the septic animals. The increased glucose uptake cannot be explained by the membrane content of GLUT1 and GLUT4 protein. Thus, during hypermetabolic sepsis, increased glucose utilization by adipose tissue is dependent on alternative processes.     

Characterization and expression pattern analysis of the facilitative glucose transporter 10 gene (slc2a10) in Danio rerio

The SLC2A10 gene located on chromosome 20q13.1 encodes the facilitative glucose transporter 10 (GLUT10), a class III member of the SLC2A facilitative glucose transporter family. Mutations in the human SLC2A10 gene cause arterial tortuosity syndrome (ATS), a rare autosomal recessive connective tissue disorder. In this work, we report the characterization of the slc2a10 ortholog gene in zebrafish (Danio rerio) and its expression pattern during embryonic development and in adult tissues. The slc2a10 gene consists of 5 exons, spanning 8 kb and mapping to a region on chromosome 11 that exhibits conserved synteny with human chromosome 20. The gene encodes Glut10, a 513 amino acid protein that maintains the 12 transmembrane domain structure typical of the GLUTs family, and shares the specific functional motifs involved in sugar transport with the vertebrate GLUT10. RT-PCR analysis showed that two specific splice variants, both including the 5’-UTR region, were expressed during embryogenesis and in different adult zebrafish tissues and organs. In situ hybridization analyses demonstrated a maternal origin of the total slc2a10 mRNA and its ubiquitous distribution until the early somitogenesis stage. In later embryonic stages, slc2a10 mRNA was detected in the otic vesicles, hatching gland cells, pectoral fin, posterior tectum and swim bladder. Overall, these results suggest a wide role of slc2a10 during zebrafish development.     

Cytokine regulation of facilitated glucose transport in human articular chondrocytes.

Glucose serves as the major energy substrate and the main precursor for the synthesis of glycosaminoglycans in chondrocytes. Facilitated glucose transport represents the first rate-limiting step in glucose metabolism. This study examines molecular regulation of facilitated glucose transport in normal human articular chondrocytes by proinflammatory cytokines. IL-1beta and TNF-alpha, and to a lesser degree IL-6, accelerate facilitated glucose transport as measured by [(3)H]2-deoxyglucose uptake. IL-1beta induces an increased expression of glucose transporter (GLUT) 1 mRNA and protein, and GLUT9 mRNA. GLUT3 and GLUT8 mRNA are constitutively expressed in chondrocytes and are not regulated by IL-1beta. GLUT2 and GLUT4 mRNA are not detected in chondrocytes. IL-1beta stimulates GLUT1 protein glycosylation and plasma membrane incorporation. IL-1beta regulation of glucose transport in chondrocytes depends on protein kinase C and p38 signal transduction pathways, and does not require phosphoinositide 3-kinase, extracellular signal-related kinase, or c-Jun N-terminal kinase activation. IL-1beta-accelerated glucose transport in chondrocytes is not mediated by endogenous NO or eicosanoids. These results demonstrate that stimulation of glucose transport represents a component of the chondrocyte response to IL-1beta. Two classes of GLUTs are identified in chondrocytes, constitutively expressed GLUT3 and GLUT8, and the inducible GLUT1 and GLUT9.     

Tissue specific expression of the splice variants of the mouse vacuolar proton-translocating ATPase a4 subunit

We have identified splicing variants of the mouse a4 subunit which have the same open reading frame but have a different 5′-noncoding sequence. Further determination of the 5′-upstream region of the a4 gene in mouse indicated the presence of two first exons (exon 1a and exon 1b) which include the 5′-noncoding sequence of each variant. The mRNAs of both splicing variants (a4-I and a4-II) show a similar expression pattern in mouse kidney by in situ hybridization. However, tissue and developmental expression patterns of the variants are different. In addition to strong expression in kidney, a4-I expression was detected in heart, lung, skeletal muscle, and testis, whereas a4-II is expressed in lung, liver, and testis. During development, a4-I was expressed beginning with the early embryonic stage, but a4-II mRNA was detected from day17. These results suggest that each a4 variant has both a tissue and developmental stage specific function.     

Complex structure and regulation of expression of the rat gene for inward rectifier potassium channel Kir7.1

Genomic organization of the rat inward rectifier K(+) channel Kir7.1 was determined in an attempt to clarify how multiple species of its mRNA are generated in a tissue-specific manner and how its expression is regulated. The rat Kir7.1 gene spans >40 kilobases (kb) and consists of eight exons; the first four exons encode the 5'-untranslated region that is unusually long ( approximately 3 kb). The coding region is located in exons 5 and 6. In the testis, exon 4 is processed as four exons (4a-4d), whereas it is recognized as a single exon in the small intestine. The three major species of rat Kir7.1 mRNA (1.4, 2.2, and 3.2 kb) were found to arise from alternative usage of the two promoters and polyadenylation signals and by alternative splicing of the 5'-noncoding exons. The splicing pattern of the 5'-noncoding exons is quite complex and highly tissue-specific, suggesting that complex mechanisms may operate to regulate the Kir7.1 expression. Deletion and mutational analysis of the promoter activity indicated that the rat Kir7.1 gene is regulated by cAMP through a CCAAT element. The cAMP induction was also demonstrated using the rat follicular cell line FRTL-5 endogenously expressing Kir7.1.