We have determined which SNAREs in rat adipose cell PM might participate in SNARE complexes with proteins from GLUT4 vesicles. The VAMPs in PM of insulin-treated cells and in GLUT4-vesicles from cells in either condition, are in a form that readily forms a SNARE complex with PM t-SNAREs and NSF. Insulin appears to activate PM and/or GLUT4-vesicles so as to increase the efficiency of SNARE complex formation. Various evidence suggests that annexins play a role in subcellular membrane trafficking. Our data indicate that insulin- stimulated GLUT4 subcellular trafficking does not rely on a change in subcellular location of any of the five annexins detected in rat adipose cells. Glucose transport activity and cell surface GLUT4 have been assessed in fast- and slow-twitch muscles of obese Zucker rats. The data suggest that the reduced insulin-stimulated glucose transport capacity results directly from an inability to effectively enhance cell surface GLUT4. Isolated rat brown adipose cells have been prepared whose viability is indicated by the expected stimulation of oxygen consumption by norepinephrine and counter-regulation of this response by insulin. Studies with these cells demonstrate that although the regulation of glucose transport by insulin is qualitatively similar to that in white adipose cells, counter- regulation by adenosine and isoproterenol is at least quantitatively and may be qualitatively different. We have also investigated whether the subcellular distribution and insulin-stimulated translocation of GLUT4 are affected when GLUT4 is overexpressed in mouse skeletal muscle. The data suggest that the increase in insulin-stimulated glucose transport following overexpression of GLUT4 is limited by factors other than the PM GLUT4 concentration. Furthermore, GLUT4 overexpression is not coupled to glucose metabolic capacity. The presence and regulation of a cGI PDE in rat brown adipose cells have been investigated. The results suggest that the hormonal regulation of cGI PDE, presumably a cGIP1 isoform, is similar to that in white adipose cells. The effects of insulin on the cell surface quantities of GLUT1, GLUT3, and GLUT4 in L6 myotubes have been determined. Insulin stimulates the translocation of all three GLUT isoforms to PM from the light microsome fraction, resulting in equal molar quantities on the cell surface. To test the hypothesis the T3 increases the partitioning of GLUT1 to the cell surface in ARL 15 cells, we have quantitated cell surface GLUT1. Most of the early (6 h) stimulation of glucose transport by T3 is mediated by an increase in the partitioning of GLUT1 to PM. With more chronic T3 treatment (48 h), the enhanced surface partitioning of GLUT1 is persistent and is superimposed on an increase in total cellular GLUT1. The subcellular localization of GLUTs 1-5 in rat pancreatic islets has been studied by immunohistochemistry. Rat islets appear to take up glucose by at least three different processes and blood glucose levels could be modulated differently by the high Km GLUT2 in B-cells, the low Km GLUT3 in B-cells, and the low Km GLUT5 in a-cells.
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