2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN (TCDD)-INDUCED DOWN-REGULATION OF GLUCOSE TRANSPORTING ACTIVITIES IN MOUSE 3T3-L1 PREADIPOCYTE
The possibility that 3T3-L1 preadipocytes, while the level of its glucose uptake activity is relatively low, may offer a useful tool for studying the cause for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced “lipolytic response” was studied. It was established first, that TCDD causes reduction of glucose uptake, one of the hallmark events of the lipolytic process. Then the function of c-Src was investigated. The antisense c-src oligonucleotide decreased the inhibitory action of TCDD on glucose uptake activity in a sequence specific manner. Since antisense oligonucleotides are known to own their blocking effects to their ability to reduce translation of proteins, Western blotting analysis was performed to verify their effectiveness. As expected, the treatment of preadipocytes with antisense c-src oligonucleotide reduced c-Src in a sequence specific manner. The treatment of antisense c-src oligonucleotide alone was sufficient to diminish the inhibitory action of TCDD on glucose uptake activity in 3T3-L1 cells, indicating that c-Src is somehow involved in the action of TCDD. In a similar manner, the contribution of c-Fos was investigated using antisense c-fos oligonucleotide, since c-Fos is known to be one of the most affected proteins by c-Src activation among AP-1 members. The treatment of antisense c-fos oligonucleotide did not block the effect of TCDD on glucose uptake activity in 3T3-L1 cells. Therefore, it is unlikely that c-Fos is very important in the lipolytic signal transduction of TCDD mediated through c-Src. In order to determine the relationship between c-Src and c-Myc in the mitotic signal transduction pathway, the effect of antisense c-myc oligonucleotide was investigated. Basically the same result as antisense c-src oligonucleotide experiment was obtained thereby, suggesting the importance of c-Myc as well as c-Src in the signal transduction of TCDD. To show the effect of antisense c-myc oligonucleotide treatment, the level of c-Myc protein by Western blotting and electrophoretic gel-mobility shift assay was assessed. However, antisense c-myc oligonucleotide treatment increased the activity of c-Myc in a sequence specific manner. This may be the result of cellular compensatory response to the initial suppression of c-Myc by antisense treatment. The observation that antisense c-fos oligonucleotide could not block the effect of TCDD, indicates that this preadipocyte model is different from the adipocyte differentiation model.