Peter R. Shepherd

Glucose Transporters and Insulin Action — Implications for Insulin Resistance and Diabetes Mellitus

Insulin was discovered more than 75 years ago, but only recently have we begun to understand the mechanisms by which insulin promotes the uptake of glucose into cells. This review discusses recent ...

Distribution of GLUT3 glucose transporter protein in human tissues

To investigate the tissue distribution of the GLUT3 glucose transporter isoform in human tissue we produced affinity purified antibodies to the COOH terminus of the human GLUT3. Both antibodies recognize a specific GLUT3 band in oocytes injected with GLUT3 mRNA but not in those injected with H2O or GLUT1, 2, 4, 5 mRNA. This immunoreactive band in GLUT3 injected oocytes is photolabelled by cytochalasin-B in the presence of L- but not D-glucose indicating that it is a glucose transporter. A high cross reactivity between the human GLUT3 antibodies and a 43 kDa cytoskeletal actin band was identified in all oocyte lysates and many human tissues. However, the specific GLUT3 band could be distinguished from the actin band by carbonate treatment which preferentially solubilized the actin band. Using these antibodies we show that GLUT3 is present as a 45-48 kDa protein in human brain with lower levels detectable in heart, placenta, liver and a barely detectable level in kidney. No GLUT3 was detected in membranes from any of 3 skeletal muscle groups investigated. We conclude that a major role of GLUT3 in humans is as the brain neuronal glucose transporter.

Human Small Intestine Facilitative Fructose/Glucose Transporter (GLUT5) Is Also Present in Insulin-Responsive Tissues and Brain: Investigation of Biochemical Characteristics and Translocation

A recent study by C.F. Burant et al. (13) demonstrates that GLUT5 is a high-affinity fructose transporter with a much lower capacity to transport glucose. To characterize the potential role of GLUT5 in fructose and glucose transport in insulin-sensitive tissues, we investigated the distribution and insulin-stimulated translocation of the GLUTS protein in human tissues by immunoblotting with an antibody to the COOH-terminus of the human GLUTS sequence. GLUTS was detected in postnuclear membranes from the small intestine, kidney, heart, four different skeletal muscle groups, and the brain, and in plasma membranes from adipocytes. Cytochalasin-B photolabeled a 53,000-Mr protein in small intestine membranes that was immunoprecipitated by the GLUT5 antibody; labeling was inhibited by D- but not L-glucose. N-glycanase treatment resulted in a band of 45,000 Mr in all tissues. Plasma membranes were prepared from isolated adipocytes from 5 nonobese and 4 obese subjects. Incubation of adipocytes from either group with 7 nM insulin did not recruit GLUT5 to the plasma membrane, in spite of a 54% insulin-stimulated increase in GLUT4 in nonobese subjects. Thus, GLUT5 appears to be a constitutive sugar transporter that is expressed in many tissues. Further studies are needed to define its overall contribution to fructose and glucose transport in insulin-responsive tissues and brain.

Expression of the brain-type glucose transporter is restricted to brain and neuronal cells in mice

SummaryNorthern blot analysis of human tissues has demonstrated the expression of the brain-type glucose transporter isoform (GLUT 3) in liver, muscle and fat, raising the possibility that this transporter isoform may play a role in the regulation of glucose disposal in these tissues in response to insulin. We have raised an anti-peptide antibody against the C-terminal 13 amino acids of the murine homologue of this transporter isoform, and determined its tissue distribution in mouse tissues and murine-derived cell lines. The antibodies recognise a glycoprotein of about 50 kilodaltons, expressed at high levels in murine brain. In contrast to human tissues, the expression of GLUT 3 in mice is restricted to the brain, and no immunoreactivity was observed in either liver, fat or muscle membranes, or in murine 3T3-L1 fibroblasts or adipocytes. In contrast, high levels of expression of this isoform were observed in the NG 108 neuroblastoma x glioma cell line, a hybrid cell derived from rat glioma and mouse neuroblastoma cells. Taken together, these data suggest that the expression of GLUT 3 in rodents is restricted to non-insulin responsive neuronal cells and hence it is likely that the factors regulating the expression of this transporter in rodents differ to those in humans.

Over-expression of GLUT4 selectively in adipose tissue in transgenic mice: Implications for nutrient partitioning

In summary, over-expression of GLUT4 selectively in fat causes increased flux of glucose into adipocytes and leads to increases in either the replication of immature pre-adipocytes or their differentiation into mature adipocytes resulting in an increase in fat cell number. This is the first model in which obesity is accounted for entirely by adipocyte hyperplasia and, therefore, is useful for studying the mechanisms involved in controlling fat cell number in vivo. GLUT4 over-expression in adipocytes of transgenic animals also increased whole- body insulin sensitivity. However, GLUT4 over-expression exclusively in adipocytes did not protect them from insulin resistance in vivo induced by high-fat feeding, in spite of the fact that insulin resistance was prevented at the level of the adipocyte. Interestingly, GLUT4 over-expression in fat protected the animals from developing further obesity when fed on a high-fat diet. It is possible that this failure to increase adiposity further is due to enhanced partitioning of glucose into fat, which may result in decreased glucose supply to muscle. This in turn may cause diversion of lipid to muscle to be oxidized as fatty acid. This diversion of lipid could result in protection against increased fat deposition in adipocytes. Further studies will be required in order to understand the molecular mechanisms by which GLUT4 over-expression in adipose tissues affects nutrient partitioning between muscle and adipose tissue and what the consequences of this are for whole-body fuel metabolism.