Stefan Schmidt

GLUT8, the enigmatic intracellular hexose transporter.

GLUT8 is a class III sugar transporter predominantly expressed in testis and brain. In contrast to the class I and class II transporters, hydrophobicity plots predict a short extracellular loop between transmembrane domain (TM)1 and TM2 and a long extracellular loop between TM9 and TM10 that contains the only N-glycosylation site. In vitro translated GLUT8 migrates as a 35-kDa protein that is glycosylated in the presence of microsomal membranes. In heterologous expression systems, glucose transport activity (Km of 2 mM) was inhibited by fructose and galactose. The transporter carries an NH2-terminal endosomal/lysosomal targeting motif ([DE]XXXL[LI]). Accordingly, constitutive GLUT8 has been found to be associated with endosomes and lysosomes but also with membranes of the endoplasmic reticulum. A similar distribution was detected after overexpression of wild-type or tagged GLUT8 in different cell systems. In these cells, none of the conventional signals tested induced a translocation of GLUT8 to the plasma membrane. Therefore, GLUT8 appears to catalyze transport of sugars or sugar derivatives through intracellular membranes. Slc2a8 knockout mice were viable, developed normally, and showed mild alterations in brain (increased proliferation of neuronal cells in dentate gyrus of the hippocampus, hyperactivity), heart (impaired transmission of electrical wave through the atrium), and sperm cells (reduced number of motile sperm cells associated with reduced mitochondrial membrane potential and ATP levels in sperm). The links between molecular function, cellular localization and phenotype of the knockout mouse is unclear and remains to be determined.

Characterization of the human SLC2A11 (GLUT11) gene: alternative promoter usage, function, expression, and subcellular distribution of three isoforms, and lack of mouse orthologue

GLUT11 (SLC2A11) is a class II sugar transport facilitator which exhibits highest similarity with the fructose transporter GLUT5 (about 42%). Here we demonstrate that separate exons 1 (exon 1A, exon 1B, and exon 1C) of the SLC2A11 gene generate mRNAs of three GLUT11 variants (GLUT11-A, GLUT11-B, and GLUT11-C) that differ in the amino acid sequence of their N-termini. All three 5′-flanking regions of exon 1A, exon 1B and exon 1C exhibited promoter activity when expressed as luciferase fusion constructs in COS-7 cells. 5′-RACE-PCR, quantitative real-time PCR, and Northern blot analysis performed with specific probes for exon 1A, 1B and 1C demonstrated that GLUT11-A is expressed in heart, skeletal muscle, and kidney, GLUT11-B in kidney, adipose tissue, and placenta, and GLUT11-C in adipose tissue, heart, skeletal muscle, and pancreas. Surprisingly, mice and rats lack the SLC2A11 gene. When expressed in Xenopus oocytes, all three GLUT11 isoforms transport glucose and fructose but not galactose. There was no apparent difference in the subcellular distribution of the three isoforms expressed in COS-7 cells. Our data indicate that different promoters and splicing of the human SLC2A11 gene generate three GLUT11 isoforms which are expressed in a tissue specific manner but do not appear to differ in their functional characteristics.

Development of diabetes in obese, insulin-resistant mice: essential role of dietary carbohydrate in beta cell destruction

Aims/hypothesisThe role of dietary carbohydrate in the pathogenesis of type 2 diabetes is still a subject of controversial debate. Here we analysed the effects of diets with and without carbohydrate on obesity, insulin resistance and development of beta cell failure in the obese, diabetes-prone New Zealand Obese (NZO) mouse.Materials and methodsNZO mice were kept on a standard diet (4% [w/w] fat, 51% carbohydrate, 19% protein), a high-fat diet (15, 47 and 17%, respectively) and a carbohydrate-free diet in which carbohydrate was exchanged for fat (68 and 20%, respectively). Body composition and blood glucose were measured over a period of 22xa0weeks. Glucose tolerance tests and euglycaemic-hyperinsulinaemic clamps were performed to analyse insulin sensitivity. Islet morphology was assessed by immunohistochemistry.ResultsMice on carbohydrate-containing standard or high-fat diets developed severe diabetes (blood glucose >16.6xa0mmol/l, glucosuria) due to selective destruction of pancreatic beta cells associated with severe loss of immunoreactivity of insulin, glucose transporter 2 (GLUT2) and musculoaponeurotic fibrosarcoma oncogene homologue A (MafA). In contrast, mice on the carbohydrate-free diet remained normoglycaemic and exhibited hyperplastic islets in spite of a morbid obesity associated with severe insulin resistance and a massive accumulation of macrophages in adipose tissue.Conclusions/interpretationThese data indicate that the combination of obesity, insulin resistance and the inflammatory response of adipose tissue are insufficient to cause beta cell destruction in the absence of dietary carbohydrate.

Targeted disruption of Slc2a8 (GLUT8) reduces motility and mitochondrial potential of spermatozoa.

GLUT8 is a class 3 sugar transport facilitator which is predominantly expressed in testis and also detected in brain, heart, skeletal muscle, adipose tissue, adrenal gland, and liver. Since its physiological function in these tissues is unknown, we generated a Slc2a8 null mouse and characterized its phenotype. Slc2a8 knockout mice appeared healthy and exhibited normal growth, body weight development and glycemic control, indicating that GLUT8 does not play a significant role for maintenance of whole body glucose homeostasis. However, analysis of the offspring distribution of heterozygous mating indicated a lower number of Slc2a8 knockout offspring (30.5:47.3:22.1%, Slc2a8+/+, Slc2a8+/−, and Slc2a8−/− mice, respectively) resulting in a deviation (p=0.0024) from the expected Mendelian distribution. This difference was associated with lower ATP levels, a reduced mitochondrial membrane potential and a significant reduction of sperm motility of the Slc2a8 knockout in comparison to wild-type spermatozoa. In contrast, number and survival rate of spermatozoa were not altered. These data indicate that GLUT8 plays an important role in the energy metabolism of sperm cells.

Deletion of Glucose Transporter GLUT8 in Mice Increases Locomotor Activity

Transport of glucose into neuronal cells is predominantly mediated by the glucose transporters GLUT1 and GLUT3. In addition, GLUT8 is expressed in some regions of the brain. By inxa0situ hybridization we detected GLUT8-mRNA in hippocampus, thalamus, and cortex. However, its cellular and physiological function is still unknown. Thus, GLUT8 knockout (Slc2a8−/−) mice were used for a screening approach in the modified hole board (mHB) behavioral test to analyze the role of GLUT8 in the central nervous system. Slc2a8−/− mice showed increased mean velocity, total distance traveled and performed more turns in the mHB test. This hyperactivity of Slc2a8−/− mice was confirmed by monitoring locomotor activity in the home cage and voluntary activity in a running wheel. In addition, Slc2a8−/− mice showed increased arousal as indicated by elevated defecation, reduced latency to the first defecation and a tendency to altered grooming. Furthermore, the mHB test gave evidence that Slc2a8−/− mice exhibit a reduced risk assessment because they performed less rearings in an unprotected area and showed significantly reduced latency to stretched body posture. Our data suggest that behavioral alterations of Slc2a8−/− mice are due to dysfunctions in neuronal processes presumably as a consequence of defects in the glucose metabolism.

Neuronal functions, feeding behavior, and energy balance in Slc2a3+/– mice

Homozygous deletion of the gene of the neuronal glucose transporter GLUT3 (Slc2a3) in mice results in embryonic lethality, whereas heterozygotes (Slc2a3+/-) are viable. Here, we describe the characterization of heterozygous mice with regard to neuronal function, glucose homeostasis, and, since GLUT3 might be a component of the neuronal glucose-sensing mechanism, food intake and energy balance. Levels of GLUT3 mRNA and protein in brain were reduced by 50% in Slc2a3+/- mice. Electrographic features examined by electroencephalographic recordings give evidence for slightly but significantly enhanced cerebrocortical activity in Slc2a3+/- mice. In addition, Slc2a3+/- mice were slightly more sensitive to an acoustic startle stimulus (elevated startle amplitude and reduced prepulse inhibition). However, systemic behavioral testing revealed no other functional abnormalities, e.g., in coordination, reflexes, motor abilities, anxiety, learning, and memory. Furthermore, no differences in body weight, blood glucose, and insulin levels were detected between wild-type and Slc2a3+/- littermates. Food intake as monitored randomly or after intracerebroventricular administration of 2-deoxyglucose or d-glucose, or food choice for carbohydrates/fat was not affected in Slc2a3+/- mice. Taken together, our data indicate that, in contrast to Slc2a1, a single allele of Slc2a3 is sufficient for maintenance of neuronal energy supply, motor abilities, learning and memory, and feeding behavior.

Essential role of glucose transporter GLUT3 for post-implantation embryonic development

Deletion of glucose transporter gene Slc2a3 (GLUT3) has previously been reported to result in embryonic lethality. Here, we define the exact time point of growth arrest and subsequent death of the embryo. Slc2a3−/− morulae and blastocysts developed normally, implanted in vivo, and formed egg-cylinder-stage embryos that appeared normal until day 6·0. At day 6·5, apoptosis was detected in the ectodermal cells of Slc2a3−/− embryos resulting in severe disorganization and growth retardation at day 7·5 and complete loss of embryos at day 12·5. GLUT3 was detected in placental cone, in the visceral ectoderm and in the mesoderm of 7·5-day-old wild-type embryos. Our data indicate that GLUT3 is essential for the development of early post-implanted embryos.

Lysosomal localization of GLUT8 in the testis – the EXXXLL motif of GLUT8 is sufficient for its intracellular sorting via AP1- and AP2-mediated interaction

The class III sugar transport facilitator GLUT8 co‐localizes with the lysosomal protein LAMP1 in heterologous expression systems. GLUT8 carries a [D/E]XXXL[L/I]‐type dileucine sorting signal that has been postulated to retain the protein in an endosomal/lysosomal compartment via interactions with clathrin adaptor protein (AP) complexes. However, contradictory findings have been described regarding the subcellular localization of the endogenous GLUT8 and the adaptor proteins that interact with its dileucine motif. Here we demonstrate that endogenous GLUT8 is localized in a late endosomal/lysosomal compartment of spermatocytes and spermatids, and that the adaptor complexes AP1 and AP2, but not AP3 or AP4, interact with its N‐terminal intracellular domain (NICD). In addition, fusion of the GLUT8 NICD to the tailless lumenal domain of the IL‐2 receptor alpha chain (TAC) protein (interleukin‐2 receptor α chain) targeted the protein to intracellular membranes, indicating that its N‐terminal dileucine signal is sufficient for endosomal/lysosomal targeting of the transporter. The localization and targeting of GLUT8 show striking similarities to sorting mechanisms reported for lysosomal proteins. Therefore, we suggest a potential role for GLUT8 in the so far unexplored substrate transport across intracellular membranes.