SKIN PENETRATION ENHANCEMENT BY TAT-GKH 487 retention time was 4.5 min for.GKH. The retention time was 12.3 min for TAT-GKH. The temperature of the column was kept at 40øC. STATISTICAL ANALYSIS Values are given as means + SEM of (n) separate experiments. Differences in value were statistically analyzed using a two-tailed two-sample t-test. RESULT AND DISCUSSION IN VITRO LIPOLYSIS IN CULTURED ADIPOCYTES Due to the fusion of TAT into GKH, it was necessary to confirm whether TAT-GKH, like GKH, still had the same lipolytic effects or not. To compare the lipolysis effects of TAT-GKH with those ofGKH, 3T3-L1 differentiated adipocytes were incubated in the presence and absence of various doses of TAT-GKH and GKH at 37øC for 2 h. Lipolysis in 3T3-L1 differentiated adipocytes was estimated by determining the amount of glyc- erol released into the medium as a result of lipolysis on triglyceride. The lipolytic agent isoproterenol was used as the positive control to confirm the absence of technical errors in this experiment and induced high lipolytic effects (data not shown). The effects of increasing concentrations of TAT-GKH and of GKH on glycerol production in adipo- cytes are depicted in Figure 1. TAT-GKH induced approximately 37.6% maximal lipolysis at 10 -5 M, compared with basal lipolysis. Both TAT-GKH and GKH repre- sented similar lipolytic effects at the same concentration and also induced lipolytic effects in a dose-dependent fashion. IN VITRO LIPOLYSIS IN ADIPOCYTES ISOLATED FROM RATS 3T3-L1 differentiated adipocytes contain considerable triglyceride in much smaller lipid storage droplets than mature primary adipocytes, which contain large unilocular lipid 6O ,-- 5O ß 30 • 20 o '10 10-4 10-• 10-• 10-7 [] GKH ß TAT-GKH Concentration (mol/L) Figure 1. Lipolytic effects of TAT-GKH in 3T3-L1 differentiated adipocytes, compared with those of GKH. Values are mean _+ SEM (n = 6), expressed as percent (%) vs basal lipolysis for glycerol and are significantly different from those for basal lipolysis.

488 JOURNAL OF COSMETIC SCIENCE droplets (20). Long-term culture with insulin to differentiate the preadipocytes increases basal lipolysis (21), and lipolytic effects could be underestimated in 3T3-L1 differenti- ated adipocytes. To identify the possibility of underestimation of GKH lipolytic effects by the use of different cell lines, adipocytes from rats were isolated to measure the lipolytic effects. TAT-GKH and GKH induced approximately 41.5% and 36.3% maximal lipolytic effects at 10 -5 M (0.77 + 0.01 and 0.74 + 0.04 glycerol pmol/lipid mg/h), respectively (Table I). TAT-GKH showed identical pattern lipolytic effects at the same concentra- tions of GKH in epididymal adipocytes isolated from rats. In addition, the lipolytic effects of TAT-GKH and those of GKH in isolated adipocytes showed the same patterns as in 3T3-L1 differentiated adipocytes (Figure 2). These results suggest that the GKH lipolytic effects were not underestimated in both cultured adipocytes and isolated adi- pocytes. Unlike a previous Leroux study (12) that the lipolytic effects of Pal-GKH and GKH in human isolated adipocytes represented 31.6% and 34.9% at 3 x 10 6 M concentration, respectively, our results showed the lipolytic effects in higher concentration (10 -5 M) than in Leroux's study. This may be due to the fact that GKH originated from human Table I Maximal Lipolytic Effects of TAT-GKH in Epididymal Adipocytes From Rats Compared With Those of GKH and Isoproterenol Lipolytic agents Glycerol (pmol/lipid mg/h) Basal lipolysis 0.54 _+ 0.01 Isoproterenol (10 6 M) 5.74 _+ 0.64 GKH (10 -5 M) 0.74 _+ 0.04* TAT-GKH (10 M) 0.77 _+ 0.01' Values are mean + SEM (n = 6). * P 0.01 when compared to values obtained with basal lipolysis. 60 .-. 50 ß .• 40 "• 3o o • 2o o •10 10 • 10 3 10 • 10 -7 [] GKH ß " TAT-GKH Concentration (mol/L) Figure 2. Lipolytic effects of TAT-GKH in epididymal adipocytes from rats compared with those of GKH. Values are mean + SEM (n = 6), expressed as percent (%) vs basal lipolysis for glycerol and are significantly different from those for basal lipolysis.