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S-2-1 Effects of insulin on membrane lipid compositions and distribution of newly synthesized fatty acids into triacylglycerols and phospholipids in rat adipocytes
S-2-1 EFFECTS OF INSULIN ON MEMBRANE LIPID COMPOSITIONS AND DISTRIBUTION OF NEWLY SYNTHESIZED FATTY ACIDS INTO TRIACYLGLYCEROLS AND PHOSPHOLIPIDS IN RAT ADIPOCYTES L.T. Ho, M.T.Kou, J.C. Perng, T. Ho, M.F. Wang and M.S. Shiao Department of Medicine, Veterans General Hospital, China The changes in lipogenesis, incorporation of U-14C-glucose into glycerol and fatty acid moieties of triacylglycerols (TG) and membrane phospholipids (PL), and fatty acid patterns of PL were elucidated in rat adipocytes in the absence or presence of insulin. Our results show that the membrane PL from fasting animals contained higher percentages of unsaturated fatty acids than those in controls. Insulin increased the contents of saturated fatty acids in TG and PL to different extents. High carbohydrate fat-free diet induced the incorporation of U- 14C-glucose label into PL and YG in opposite directions. CONTROL -insulin +insulin FASTING -insulin +insulin
Fatty acid patterns of PL in adipocyte plasma C12:0 C16:0 C18:0 C18:1 C18:2 15.4±1.9 16.310.4 15.310.4 ii.410.3 21.410.3 8.3±1.2 19.710.5 19.310.6 13.710.5 24.810.2
membrane C20:4 20.1±1.0 14.1±1.0
21.211.8 ii.8iO.4 i0.510.4 9.010.3 23.410.3 24.1±1.7 17.9+__2.2 12.710.6 6.P+1.2 II.4+P.2 16.6+1.7 24.810.5
S-2-2 STUDIES WITH SITE-SPECIFIC ANTIBODIES SUGGEST THE TISSUE DIFFERENCE OF GLUCOSE TRANSPORTER PROTEIN. Y. Oka, T. Asano, M. Kasuga, Y. Kanazawa and F. Takaku The Third Department of Internal Medicine, University of Tokyo School of Medicine, Tokyo, Japan Three site-specific antibodies(Ab) were prepared by immunizing rabbits with synthesized short peptides derived from deduced amino acid sequence of human hepatoma HepG2 glucose transporter(GT) and rat brain GT, and further a~finity-purified. Virtually all the human RBC GT photoaffinity-labeled with (~H)-cytochalasin B was immunoprecipitated by Ab to a mid portion of GT(AbM), Ab to a domain close to C-terminus, or Ab to C-terminus. These domains are common in human and rat GT. Interestingly, only AbM inhibited cytochalasin B labeling and glucose transport activity in reconstituted vesicles when solubilized RBC membranes were preincubated with Ab. Further, all three Ab recognized the rat brain protein of Mr 50,000 on immunoblots. The same molecular weight protein was labeled with cytochalasin B and the labeling was inhibited by the presence of D-glucose, indicating that this protein is GT. However, only 20-30% of the labeled rat brain GT was immunoprecipitated by Ab. Surprisingfy, none of these Ab recognized rat adipocyte GT either by immunoprecipitation or immunoblotting method. Furthermore, human adipocyte GT and RBC GT were trypsinized in a different fashion. These data suggest (1) human RBC GT is very similar to human HepG2 GT, (2) cytochalasin B binding region resides close to the mid portion of human RBC GT, (3) rat brain contains more than two types of GT, (4) considerable portions of adipocyte GT appear to be different from human RBC GT.
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Fatty acid patterns of PL in adipocyte plasma C12:0 C16:0 C18:0 C18:1 C18:2 15.4±1.9 16.310.4 15.310.4 ii.410.3 21.410.3 8.3±1.2 19.710.5 19.310.6 13.710.5 24.810.2
membrane C20:4 20.1±1.0 14.1±1.0
21.211.8 ii.8iO.4 i0.510.4 9.010.3 23.410.3 24.1±1.7 17.9+__2.2 12.710.6 6.P+1.2 II.4+P.2 16.6+1.7 24.810.5
S-2-2 STUDIES WITH SITE-SPECIFIC ANTIBODIES SUGGEST THE TISSUE DIFFERENCE OF GLUCOSE TRANSPORTER PROTEIN. Y. Oka, T. Asano, M. Kasuga, Y. Kanazawa and F. Takaku The Third Department of Internal Medicine, University of Tokyo School of Medicine, Tokyo, Japan Three site-specific antibodies(Ab) were prepared by immunizing rabbits with synthesized short peptides derived from deduced amino acid sequence of human hepatoma HepG2 glucose transporter(GT) and rat brain GT, and further a~finity-purified. Virtually all the human RBC GT photoaffinity-labeled with (~H)-cytochalasin B was immunoprecipitated by Ab to a mid portion of GT(AbM), Ab to a domain close to C-terminus, or Ab to C-terminus. These domains are common in human and rat GT. Interestingly, only AbM inhibited cytochalasin B labeling and glucose transport activity in reconstituted vesicles when solubilized RBC membranes were preincubated with Ab. Further, all three Ab recognized the rat brain protein of Mr 50,000 on immunoblots. The same molecular weight protein was labeled with cytochalasin B and the labeling was inhibited by the presence of D-glucose, indicating that this protein is GT. However, only 20-30% of the labeled rat brain GT was immunoprecipitated by Ab. Surprisingfy, none of these Ab recognized rat adipocyte GT either by immunoprecipitation or immunoblotting method. Furthermore, human adipocyte GT and RBC GT were trypsinized in a different fashion. These data suggest (1) human RBC GT is very similar to human HepG2 GT, (2) cytochalasin B binding region resides close to the mid portion of human RBC GT, (3) rat brain contains more than two types of GT, (4) considerable portions of adipocyte GT appear to be different from human RBC GT.
SlO