The authors declare no conflicts of interest.
Acknowledgments
The authors are grateful to the National Science Council of Taiwan for the grants to H.
L. Cheng (NSC 99-2313-B-020-003-MY3; NSC 2317-B-020-003), to C. I. Chang (NSC 97-2317-B-020 -002), and to C. H. Chou (NSC 96-2317-B-039-002).
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Figure Captions
Fig. 1 - Animal tests to assess the hypoglycaemic activity of ME in STZ-induced diabetic mice.
A, B, and C, diabetic mice administered with 100 mg/kg/d of ME, 50 mg/kg/d of ME, or the vehicle, respectively. D, normal mice administered with the vehicle. On the first, third, fifth, and sixth day of administration, blood glucose was measured before (0 h) and 2 h after the
administration. Data represent mean ± standard error of the mean (SEM; N = 6). *P < 0.05 versus 0-h blood glucose of the respective day; #P < 0.05 versus 0-h blood glucose of Day 1.
Fig. 2 - Glucose uptake assays for insulin-resistant cells treated with partitions or fractions of ME. A, a flowchart showing the partitioning and further isolation of compounds of the methanol extract of C. moschata. B, C, and D, glucose uptake assays performed using insulin-resistant cells. FL83B cells were treated with TNF- for 5 h to induce insulin resistance (Groups 3-8), followed by treatment with insulin and the indicated partition or fraction for another 5 h (Groups 4-8), during which glucose concentration in the culture medium was monitored to calculate the glucose consumption of the cells. The glucose uptake relative to Group 1 was plotted as the histograms. Data represent the mean ± standard deviation (SD) of triplicate. B, assays for cells treated with 100 g/mL of ME, EA, Bu, or W. C, assays for cells treated with 1 g/mL of fraction 1. D, assays for cells treated with 1 g/mL of fractions 2-23, respectively. *P < 0.05 against Group 3 by two-way ANOVA.
Fig. 3 - The hypoglycaemic activities in vivo and constituents of F19 and F22. A to C, STZ-induced diabetic mice were administered with 200 mg/kg/d of F19 (A), 200 mg/kg/d of F22 (B), or the vehicle (C). D, normal mice administered with the vehicle. On the first, second and third day of administration, blood glucose was measured before (0 h) and 2 h after the administration.
Data represent mean ± SEM (N = 6). *P < 0.05 versus 0-h blood glucose of the respective day;
#P < 0.05 versus 0-h blood glucose of Day 1. E, the structures of compounds isolated from F19 and F22. Compound 8 was isolated from F22. The others were isolated from F19.
Fig. 4 - Glucose uptake and cytotoxicity assays for compounds 1-10. A, glucose uptake assays in normal FL83B cells. Cells were treated with the vehicle (control), 100 nM of insulin (Ins), or 20
M of the indicated compound (1-10) for 5 h, during which glucose concentration in the culture medium was monitored to determine the glucose uptake of the cells. The glucose uptake relative to control was plotted as the histograms. Data represent the mean ± SD of triplicate experiments.
*P < 0.05 versus control. B, glucose uptake assays as in A, but the concentration of the indicated compound (1-8) was 40 M. C, glucose uptake assays in insulin-resistant cells. FL83B cells were treated with TNF- for 5 h to result in insulin resistance (Groups 3-8), followed by treatment with insulin and the indicated compound for another 5 h (Groups 4-8). The glucose uptake relative to Group 1 was plotted as the histograms. Data represent the mean ± SD of triplicate experiments. *P < 0.05 versus Group 3. D, cytotoxicity assays of 4, 8, 9, and 10 in FL83B cells.
Cells were treated with 0 (control), 2, 20, 30, or 50M of the compound for 24 h. The percentage of viable cells relative to control was calculated. Data represent the mean ± SD of triplicate experiments, and were statistically analyzed against the control by one-way ANOVA. None of them showed a significant difference.
Fig. 5 - Characterization of the insulin-like activities of 9 and 10 in normal cells. A, B, and C, Western blotting of the levels of tyrosine phosphorylated and total IRS-1 (A, IRS-Y-P and IRS), phosphorylated and total Akt (B, P-Akt and Akt), as well as phosphorylated and total AMPK (C, P-AMPK and AMPK) in FL83B cells, respectively. These were treated with insulin (Lanes 2 and 7), 2, 20, or 30 M of 9 (Lanes 3, 4, and 5) or 10 (Lanes 8, 9, and 10) for 30 min. Band intensities relative to Lane 1 or Lane 6 in each blot were determined after normalization by the
corresponding total protein and plotted as the respective histogram. A and B were performed twice independently, the blots were from one of the experiments and the histograms show the mean of the two experiments. D, analysis by Western blot of the levels of phosphorylated and total AS160 (P-AS160 and AS160), AMPK, and ACC-1 (P-ACC-1 and ACC-1) in FL83B cells,
which were treated with the vehicle (Lanes 1 and 3), insulin (Lane 2), 50 M of troglitazone (TZD; Lanes 4 and 5), 20 M of 9 (Lanes 6 and 7), or 20 M of 10 (Lanes 8 and 9) for 30 min. In Lanes 3, 5, 7, and 9, 30 M of Compound C (cpd C) was added 2 h before the addition of the indicated compound. Experiments were repeated 3 times independently. The blots shown were the results of one of the experiments. Band intensities relative to Lane 1 in each blot were determined after normalization by the corresponding total protein, and the mean ± SD of the 3 experiments was plotted as the histograms. E, glucose uptake assays in normal FL83B cells as in Fig. 4A. The cells were treated with the vehicle (Groups 1, 2, 7, and 8), insulin (Groups 3, 4, 9, and 10), 9 (Groups 5 and 6), or 10 (Groups 11 and 12) for 5 h. In Groups 2, 4, 6, 8, 10, and 12, 30
M of Compound C was added 2 h before the addition of the indicated chemical. The glucose uptake relative to Group 1 (the left histogram) or Group 7 (the right histogram) is presented. Data represent the mean ± SD of triplicate experiments. *P < 0.05 versus Group 1 or Group 7; #P <
0.05 between the indicated groups.
Fig. 6 - Characterization of the hypoglycaemic activities of 4, 8, and 9 in insulin-resistant cells.
Analysis by Western blot of the levels of phosphorylated and total Akt (P-Akt and Akt, Lanes 1-8), and those of AS160 (P-AS160 and AS160, Lanes 9-16) in FL83B cells. The cells were treated with TNF-(Lanes 3-8 and 11-16) for 5 h to induce insulin resistance, followed by treatment with 50 M troglitazone (TZD; Lanes 5, 6, 13, and 14), 20 M 4 (A, Lanes 7, 8, 15, and 16), 20
M 8 (B, Lanes 7, 8, 15, and 16), or 20 M 9 (C, Lanes 7, 8, 15, and 16) for 30 min. In Lanes 2, 4, 6, 8, 10, 12, 14, and 16, 100 nM of insulin was added simultaneously with the indicated compound. Experiments were repeated twice independently. The blots shown were from one of the experiments. Band intensities relative to Lane 1 or Lane 9 in each blot were determined after normalization by total Akt or AS160, and the mean of the 2 experiments was plotted as the respective histogram.