Adaptation to glucose deprivation in 3T3-L1 adipocytes

The phenomenon of glucose transport activation in response to glucose deprivation was studied in 3T3-L1 adipocytes. This effect is dependent on new protein synthesis. We carried out glucose deprivation in the presence of fructose, which provided cells with a source of carbohydrate and interfered little with the observed 8- to 10-fold transport activation. A novel procedure for the subfraction of 3T3-L1 adipocytes was used to rule out the involvement of translocation in transport activation. We established this protocol to study the subcellular distribution of the two glucose transporters present in 3T3-L1 adipocytes, GLUT1 and GLUT4. GLUT1 was shown to be the major transporter at the cell surface. Glucose deprivation did not alter the level of either transporter at the cell surface, with 20% of GLUT1 and 3% of GLUT4 appearing at the plasma membrane in both control and glucose-deprived cells. We also showed that plasma membranes of glucose-deprived cells retained transport activity. Plasma membrane proteins were then analyzed using the techniques of cell surface biotinylation and our novel subfractionation procedure. In addition, newly synthesized proteins from total membrane preparations were analyzed by two-dimensional gel electrophoresis using metabolic radiolabeling. We studied adaptation to glucose deprivation with addition of glycosylation and protein degradation inhibitors and with glucose re-feeding experiments. Tunicamycin diminished the activation response by 50%. Re-addition of glucose to deprived cells returned transport levels to basal within 4 hours. This was unaffected by inhibition of protein degradation. Transport activation was twice as fast in glucose deprived then refed cells compared with fed controls. Novel proteins were synthesized during glucose deprivation. One glucose-dependent protein (50 kDa, pI 5.2) was isolated and microsequenced which resulted in some sequence that was novel and some that matched the intermediate filament protein vimentin. Unlike vimentin which by immunoblotting was shown not to change in response to glucose deprivation, we observed that this 50 kDa protein appeared with glucose withdrawal and disappeared with glucose refeeding. This protein and at least one other which requires future study are implicated in adaptation of 3T3-L1 adipocytes to glucose deprivation.