Analysis of the glucose transporter content of islet cell lines: Implications for glucose-stimulated insulin release

Glucose transport across the plasma membrane of mammalian cells is mediated by a family of homologous proteins. Each glucose transporter isoform has a specific tissue distribution which relates to that tissue's demand for glucose. The β-cells of pancreatic islets are known to express a distinct glucose transporter isoform, termed GLUT 2, which has a high K_m for glucose. In this study, we examined the glucose transporter content of normal rat islets and three beta cell lines, β-TC, HIT and RIN cells. We show that at the protein level, GLUT 2 is the only detectable transporter isoform in normal islets, and that all three cell lines also express detectable GLUT 2. In contrast, all three cell lines expressed high levels of GLUT 1, but this isoform was not detected in normal islets. Neither the native islets nor any of the cell lines expressed GLUT 3. The insulin-responsive glucose transporter GLUT 4 was detected at very low levels in β-TC cells; to our knowledge, this is the only non-muscle or adipose cell line which expresses this isoform. We propose that the elevated level of GLUT 1 expression, together with a reduced expression of the high K_m transporter GLUT 2, may account for the characteristics aberrant patterns of glucose-stimulated insulin release in cell lines derived from β-cells.     

Glucose transporter expression in English sparrows (Passer domesticus)

Patterns of glucose transporter expression have been well-characterized in mammals. However, data for birds is currently restricted to isolated cells, domestic chickens and chicks, and ducklings. Therefore, in the present study, protein and gene expression of various glucose transporters (GLUTs) in English sparrow extensor digitorum communis, gastrocnemius and pectoralis muscles as well as heart, kidney, and brain tissues were examined. The hypothesis is that the expression pattern of avian GLUTs differs from mammals to maintain the high plasma glucose levels of birds and insulin insensitivity. Our studies failed to identify a GLUT4-like insulin responsive transporter in sparrows. GLUT1 gene expression was identified in all tissues examined and shares 88% homology with chicken and 84% homology with human GLUT1. Compared to the rat control, GLUT1 immunostaining of sparrow extensor digitorum communis muscle was weak and appeared to be localized to blood vessels whereas immunostaining of gastrocnemius muscles was comparable to rat muscle controls. Gene expression of GLUT3 was identified in all tissues examined and shares 90% gene sequence homology with chicken embryonic fibroblast and 75% homology with human GLUT3. Protein expression of GLUT3 was not determined as an avian antibody is not available. Moreover, the C-terminus of the mammalian GLUT3 transporter, against which antibodies are typically designed, differs significantly among species. The predominant difference of chicken and sparrow GLUT expression patterns from that of mammals is the lack of an avian GLUT4. The absence of this insulin responsive GLUT in birds may be a contributing factor to the observed high blood glucose levels and insulin insensitivity.     

Expression of glucose transporter 4 in the human pancreatic islet of Langerhans

Glucose transporter 4 (GLUT4) is the main insulin-responsive glucose transporter in skeletal muscle and adipose tissue of human and rodent, and is translocated to the plasma membrane in response to insulin. GLUT2 is well known as the main glucose transporter in pancreatic islets and could highly regulate glucose-stimulated insulin secretion by B-cells as a glucose sensor. We confirmed the presence of GLUT4 mRNA and GLUT4 protein in pancreas in the human. Indirect immunohistochemistry showed that the pancreatic islets of human and rat were conspicuously labeled by anti-GLUT4 antibody. The presence of placental leucine aminopeptidase (P-LAP), a homologue of insulin-regulated aminopeptidase (IRAP), was also shown in the human pancreatic islet. IRAP/P-LAP is thought to be involved in glucose metabolism. This study provides the first evidence that GLUT4 is present in human and rat pancreatic islets and may suggest its specific role in glucose homeostasis in conjunction with IRAP/P-LAP.     

Expression of human glucose transporters in Xenopus oocytes: kinetic characterization and substrate specificities of the erythrocyte, liver, and brain isoforms

We describe the functional expression of three members of the family of human facilitative glucose transporters, the erythrocyte-type transporter (GLUT 1), the liver-type transporter (GLUT 2), and the brain-type transporter (GLUT 3), by microinjection of their corresponding mRNAs into Xenopus oocytes. Expression was determined by the appearance of transport activity, as measured by the transport of 3-O-methyl-D-glucose or 2-deoxy-D-glucose. We have measured the Km for 3-O-methyl-D-glucose of GLUTs 1, 2, and 3, and the results are discussed in light of the possible roles for these different transporters in the regulation of blood glucose. The substrate specificity of these transporter isoforms has also been examined. We show that, for all transporters, the transport of 2-deoxy-D-glucose is inhibited by D-but not by L-glucose. In addition, both D-galactose and D-mannose are transported by GLUTs 1-3 at significant rates; furthermore, GLUT 2 is capable of transporting D-fructose. The nature of the glucose binding sites of GLUTs 1-3 was investigated by using hexose inhibition of 2-deoxy-D-glucose uptake. We show that the characteristics of this inhibition are different for each transporter isoform.     

Expression of K-Cl cotransporters in the α-cells of rat endocrine pancreas

The expression of K⁺-Cl⁻ cotransporters (KCC) was examined in pancreatic islet cells. mRNA for KCC1, KCC3a, KCC3b and KCC4 were identified by RT-PCR in islets isolated from rat pancreas. In immunocytochemical studies, an antibody specific for KCC1 and KCC4 revealed the expression of KCC protein in α-cells, but not pancreatic β-cells nor δ-cells. A second antibody which does not discriminate among KCC isoforms identified KCC expression in both α-cell and β-cells. Exposure of isolated α-cells to hypotonic solutions caused cell swelling was followed by a regulatory volume decrease (RVD). The RVD was blocked by 10 μM [dihydroindenyl-oxy] alkanoic acid (DIOA; a KCC inhibitor). DIOA was without effect on the RVD in β-cells. NEM (0.2 mM), a KCC activator, caused a significant decrease of α-cell volume, which was completely inhibited by DIOA. By contrast, NEM had no effects on β-cell volume. In conclusion, KCCs are expressed in pancreatic α-cells and β-cells. However, they make a significant contribution to volume homeostasis only in α-cells.     

Carboxy terminus of glucose transporter 3 contains an apical membrane targeting domain

We previously demonstrated that distinct facilitative glucose transporter isoforms display differential sorting in polarized epithelial cells. In Madin-Darby canine kidney (MDCK) cells, glucose transporter 1 and 2 (GLUT1 and GLUT2) are localized to the basolateral cell surface whereas GLUTs 3 and 5 are targeted to the apical membrane. To explore the molecular mechanisms underlying this asymmetric distribution, we analyzed the targeting of chimeric glucose transporter proteins in MDCK cells. Replacement of the carboxy-terminal cytosolic tail of GLUT1, GLUT2, or GLUT4 with that from GLUT3 resulted in apical targeting. Conversely, a GLUT3 chimera containing the cytosolic carboxy terminus of GLUT2 was sorted to the basolateral membrane. These findings are not attributable to the presence of a basolateral signal in the tails of GLUTs 1, 2, and 4 because the basolateral targeting of GLUT1 was retained in a GLUT1 chimera containing the carboxy terminus of GLUT5. In addition, we were unable to demonstrate the presence of an autonomous basolateral sorting signal in the GLUT1 tail using the low-density lipoprotein receptor as a reporter. By examining the targeting of a series of more defined GLUT1/3 chimeras, we found evidence of an apical targeting signal involving residues 473-484 (DRSGKDGVMEMN) in the carboxy tail. We conclude that the targeting of GLUT3 to the apical cell surface in MDCK cells is regulated by a unique cytosolic sorting motif.     

Cellular hypoxia of pancreatic beta-cells due to high levels of oxygen consumption for insulin secretion in vitro

Cellular oxygen consumption is a determinant of intracellular oxygen levels. Because of the high demand of mitochondrial respiration during insulin secretion, pancreatic β-cells consume large amounts of oxygen in a short time period. We examined the effect of insulin secretion on cellular oxygen tension in vitro. We confirmed that Western blotting of pimonidazole adduct was more sensitive than immunostaining for detection of cellular hypoxia in vitro and in vivo. The islets of the diabetic mice but not those of normal mice were hypoxic, especially when a high dose of glucose was loaded. In MIN6 cells, a pancreatic β-cell line, pimonidazole adduct formation and stabilization of hypoxia-inducible factor-1α (HIF-1α) were detected under mildly hypoxic conditions. Inhibition of respiration rescued the cells from becoming hypoxic. Glucose stimulation decreased cellular oxygen levels in parallel with increased insulin secretion and mitochondrial respiration. The cellular hypoxia by glucose stimulation was also observed in the isolated islets from mice. The MIN6 cells overexpressing HIF-1α were resistant to becoming hypoxic after glucose stimulation. Thus, glucose-stimulated β-cells can become hypoxic by oxygen consumption, especially when the oxygen supply is impaired.     

Glucose transporter expression in rat embryo and uterus during decidualization, implantation, and early postimplantation.

Efficient transfer of glucose from the mother to the embryonic compartment is crucial to sustain the survival and normal development of the embryo in utero, because the embryo's production of this primary substrate for oxidative metabolism is minimal. In the present study, the temporal sequence of expression of the sodium-independent facilitative glucose transporter isoforms GLUTs 1, 3, 4, and 5 was investigated in the developing rat uteroembryonic unit between conception and Gestational Day 8 using immunohistochemistry. The GLUTs 1, 3, and 4 were expressed in the embryonic tissues after the start of implantation, being colocalized in the parietal endoderm, visceral endoderm, primary ectoderm, extraembryonic ectoderm, and the ectoplacental cone. In the uterus, a faint GLUT1 labeling emerged, but not until Gestational Day 3, in the luminal epithelium, endometrial stroma, and decidual cells. The intensity of GLUT1 staining increased in the latter population with progressing decidualization. Endometrial glands and myometrial smooth muscle cells stained neither for GLUT1 nor for GLUT3 until postimplantation. During all developmental stages examined, GLUT4 was visualized throughout the pregnant rat uterus, as was GLUT3 (with the above-mentioned exceptions). The density of GLUT5 was generally less than the sensitivity of the immunohistochemical detection method in all tissues investigated. In conclusion, the data point to a significant expression of the high-affinity glucose transporters GLUTs 1, 3, and 4 in the rat uteroembryonic unit, providing supportive evidence for an important role of facilitative glucose diffusion during peri-implantation development.     

Syndecan-4 is associated with beta-cells in the pancreas and the MIN6 beta-cell line

Basement membranes (BM) in the pancreatic islet are important for islet survival and function, but supplementation of isolated islets with these components have had limited success. Currently, little is understood about which BM components and proteoglycans are essential to maintaining islet homeostasis. This study therefore aimed to characterize the BM components and proteoglycans of the islet in the mouse, rat and rabbit species. The BM of the mouse islet was varied in continuity around the islet and was discontinuous in the rat and rabbit islets. The BM consisted of collagen IV, laminin, fibronectin and perlecan in the mouse and was in tight association with the underlying islet endothelium. None of these components were found directly associated with the β-cells in tissue and in the MIN6 β-cell line. In contrast, heparan sulfate (HS) was distributed throughout the islet in all three species in a pattern distinctly different to that of perlecan and was observed mainly on the β-cells and not the α-cells in the mouse and rat. Similarly, syndecan-4 showed a staining pattern almost identical to that of HS and was mostly observed on the β-cells, not α-cells, in the mouse and rat. Both HS and syndecan-4 were also observed in the MIN6 β-cell line. The mouse islet and MIN6 syndecan-4 were both ~37?kDa in size, after deglycosylation with heparitinase. These results indicate that syndecan-4 may play an important role in β-cell function and that the cell-surface HS proteoglycans may be the missing link to maintaining islet longevity after isolation.     

SLC30A family expression in the pancreatic islets of humans and mice: cellular localization in the β-cells

Zinc is a vital co-factor for insulin metabolism in the pancreatic β-cell, involved in synthesis, maturation, and crystallization. Two families of zinc transporters, namely SLC30A (ZNT) and SLC39A (ZIP) are involved in maintaining cellular zinc homeostasis in mammalian cells. Single nuclear polymorphisms or mutations in zinc transporters have been associated with insulin resistance and risk of type 2 diabetes (T2D) in both humans and mice. Thus, mice can be useful for studying the underlying mechanisms of zinc-associated risk of T2D development. To determine potential differences in zinc transporter expression and cellular localization in the pancreatic β-cells between humans and mice, we examined all members (ZNT1-10) of the ZNT family in pancreatic islets and in β-cell lines derived from both species using immunohistochemistry and immunofluorescence microscopic analysis. We found that there were no substantial differences in the expression of nine ZNT proteins in the human and mouse islets and β-cells with exception of ZNT3, which was only detected in human β-cells, but not in mouse β-cells. Moreover, we found that ZNT2 was localized on the cell surface of both human and mouse β-cells, suggesting a role of ZNT2 in direct export of zinc out of the β-cell. Together, our study suggests functional conservations of the ZNT proteins between humans and mice. We believe that our results are of interest for future studies in the association of zinc metabolism with risk of T2D in humans using mouse models.     

Hexose metabolism in pancreatic islets. The phosphorylation of fructose

Fructose, like glucose, rapidly equilibrates across the plasma membrane of pancreatic islet cells, but is poorly metabolized and is a weak insulin secretagogue in rat pancreatic islets. A possible explanation for such a situation was sought by investigating the modality of fructose phosphorylation in islet homogenates. Several findings indicated that the phosphorylation of fructose is catalyzed by hexokinase, but not fructokinase. First, at variance with the situation found in liver homogenates, the phosphorylation of fructose in the islet homogenate was unaffected by K+ and inhibited by glucose, mannose, glucose 6-phosphate or glucose 1,6-bisphosphate. Second, the Km for fructose was much higher in islets than in liver. Third, in islet homogenates the Km and Vmax for fructose were much higher than those for glucose or mannose phosphorylation, at low aldohexose concentrations, in good agreement with the properties of purified hexokinase. In intact islets fructose augmented the islet content in glucose 6-phosphate sufficiently to cause marked inhibition of its own rate of phosphorylation. These findings may account, in part at least, for the low rate of fructose utilization by rat pancreatic islets.     

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.