Michael Mueckler

An accelerated rate of glucose transport is among the most characteristic biochemical markers of cellular transformation. To study the molecular mechanism by which transporter activity is altered, cultured rodent fibroblasts transfected with activated myc, ras, or src oncogenes were used. In myc-transfected cells, the rate of 2-deoxy-D-glucose uptake was unchanged. However, in cells transfected with activated ras and src oncogenes, the rate of glucose uptake was markedly increased. The increased transport rate in ras- and src-transfected cells was paralleled by a marked increase in the amount of glucose transporter protein, as assessed by immunoblots, as well as by a markedly increased abundance of glucose transporter messenger RNA. Exposure of control cells to the tumor-promoting phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) for 18 hours had a similar effect of increasing the rate of glucose transport and the abundance of transporter messenger RNA. For ras, src, and TPA, the predominant mechanism responsible for activation of the transport system is increased expression of the structural gene encoding the glucose transport protein.

Insulin stimulates glucose transport largely by mediating translocation of the insulin-sensitive glucose transporter (GLUT4) from an intracellular compartment to the plasma membrane. Using single cell microinjection of 3T3-L1 adipocytes, coupled with immunofluorescence detection of GLUT4 proteins, we have determined that inhibition of endogenous p21ras or injection of oncogenic p21ras has no effect on insulin-stimulated GLUT4 translocation. On the other hand, microinjection of anti-phosphotyrosine antibodies or inhibition of endogenous phosphatidylinositol 3-kinase by microinjection of a GST-p85 SH2 fusion protein markedly inhibits this biologic effect of insulin. These data suggest that the p21ras/mitogen-activated protein kinase pathway is not involved in this metabolic effect of insulin, whereas tyrosine phosphorylation and stimulation of phosphatidylinositol 3-kinase activity are critical components of this signaling pathway. Insulin stimulates glucose transport largely by mediating translocation of the insulin-sensitive glucose transporter (GLUT4) from an intracellular compartment to the plasma membrane. Using single cell microinjection of 3T3-L1 adipocytes, coupled with immunofluorescence detection of GLUT4 proteins, we have determined that inhibition of endogenous p21ras or injection of oncogenic p21ras has no effect on insulin-stimulated GLUT4 translocation. On the other hand, microinjection of anti-phosphotyrosine antibodies or inhibition of endogenous phosphatidylinositol 3-kinase by microinjection of a GST-p85 SH2 fusion protein markedly inhibits this biologic effect of insulin. These data suggest that the p21ras/mitogen-activated protein kinase pathway is not involved in this metabolic effect of insulin, whereas tyrosine phosphorylation and stimulation of phosphatidylinositol 3-kinase activity are critical components of this signaling pathway.

The pathogenesis of many lung diseases involves neutrophilic inflammation. Neutrophil functions, in turn, are critically dependent on glucose uptake and glycolysis to supply the necessary energy to meet these functions. In this study, we determined the effects of p38 mitogen-activated protein kinase and hypoxia-inducible factor (HIF)-1, as well as their potential interaction, on the expression of membrane glucose transporters and on glucose uptake in murine neutrophils. Neutrophils were harvested and purified from C57BL/6 mice and stimulated with lipopolysaccharide (LPS) in the presence or absence of specific p38 and HIF-1 inhibitors. Glucose uptake was measured as the rate of [3H]deoxyglucose (DG) uptake. We identified GLUT-1 in mouse neutrophils, but neither GLUT-3 nor GLUT-4 were detected using Western blot analysis, even after LPS stimulation. LPS stimulation did not increase GLUT-1 protein levels but did cause translocation of GLUT-1 from the cell interior to the cell surface, together with a dose-dependent increase in [3H]DG uptake, indicating that glucose uptake is regulated in these cells. LPS also activated both p38 and the HIF-1 pathway. Inhibitors of p38 and HIF-1 blocked GLUT-1 translocation and [3H]DG uptake. These data suggest that LPS-induced increases in neutrophil glucose uptake are mediated by GLUT-1 translocation to the cell surface in response to sequential activation of neutrophil p38 and HIF-1alpha in neutrophils. Given that neutrophil function and glucose metabolism are closely linked, control of the latter may represent a new target to ameliorate the deleterious effects of neutrophils on the lungs.

To delineate the signaling pathway leading to glucose transport protein (GLUT4) translocation, we examined the effect of microinjection of the nonhydrolyzable GTP analog, guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), into 3T3-L1 adipocytes. Thirty minutes after the injection of 5 mM GTPgammaS, 40% of injected cells displayed surface GLUT4 staining indicative of GLUT4 translocation compared with 55% for insulin-treated cells and 10% in control IgG-injected cells. Treatment of the cells with the phosphatidylinositol 3-kinase inhibitor wortmannin or coinjection of GST-p85 SH2 fusion protein had no effect on GTPgammaS-mediated GLUT4 translocation. On the other hand, coinjection of antiphosphotyrosine antibodies (PY20) blocked GTPgammaS-induced GLUT4 translocation by 65%. Furthermore, microinjection of GTPgammaS led to the appearance of tyrosine-phosphorylated proteins around the periphery of the plasma membrane, as observed by immunostaining with PY20. Treatment of the cells with insulin caused a similar phosphotyrosine-staining pattern. Electroporation of GTPgammaS stimulated 2-deoxy-D-glucose transport to 70% of the extent of insulin stimulation. In addition, immunoblotting with phosphotyrosine antibodies after electroporation of GTPgammaS revealed increased tyrosine phosphorylation of several proteins, including 70- to 80-kDa and 120- to 130-kDa species. These results suggest that GTPgammaS acts upon a signaling pathway either downstream of or parallel to activation of phosphatidylinositol 3-kinase and that this pathway involves tyrosine-phosphorylated protein(s).

A strategy was developed to generate expressed sequence tags (ESTs) from human pancreatic islet gene products using differential display of mRNA. Screening of over 2,000 cDNA amplification products identified 42 cDNAs that were preferentially expressed in pancreatic islets relative to exocrine tissue. Public database analysis showed that 29 (69%) corresponded to novel genes, in contrast with only 66 of 250 (26.4%) cDNA clones randomly selected from a human islet library. Reverse transcription-polymerase chain reaction (RT-PCR) and/or Northern analysis of RNA from multiple tissues confirmed that expression was enhanced in human islet cell RNA for 11 of 15 tested cDNAs. Sequence-tagged sites developed from 19 islet cDNAs were used to map these genes to human chromosomes using a combination of monochromosomal somatic-cell hybrids, genome-wide radiation hybrids, and mega-yeast artificial chromosome analysis. These results indicate that this PCR-based cDNA selection strategy yields information on a distinct subset of pancreatic islet transcribed sequences, which complements ongoing human EST identification efforts based on random cDNA selection. These mapped ESTs may be used to assist in the positional cloning of diabetes susceptibility genes.