Fig. 1

Effect of GCSW210 on toxicity in and glucose uptake of zebrafish larvae. A) Indications of toxicity; B) survivability; C) fluorescence imaging of glucose uptake; D) level of glucose uptake. Fertilized eggs were collected and placed in 96-well culture plates. After 7 days post-fertilization (dpf), larvae were treated with GCSW210 (125 and 250 μg/mL) for 24 h post-fertilization (hpf), and mortality and malformation were observed. All values are presented as mean ± SE from three independent repeated experiments and analyzed by one-way analysis of variance followed by Dunnett test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. control. Abbreviations: GCSW210; subcritical-water extract of Gracilaria chorda (GC) at 210 °C, dpf; days post-fertilization, hpf; hours post-fertilization.

Fig. 2

Effect of HG on toxicity and glucose uptake of zebrafish larvae. A) survivability in different concentrations of glucose solution for 72 h; B) level of glucose uptake; C) fluorescence imaging of glucose uptake (2.0X). Zebrafish larvae were exposed to d-glucose solution (32, 65, and 130 mM) from 5 dpf to 8 dpf to induce IR to create a T2DM model. Data are presented as mean ± SE (n = 8) and analyzed by one-way analysis of variance followed by Dunnett test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. control. Abbreviations: HG; high glucose, T2DM; type 2 diabetes mellitus.

Fig. 3

Effect of GCSW210 on insulin sensitivity in HG-mediated insulin resistance model of zebrafish larvae. A) fluorescence imaging of glucose uptake (4.0X); B) level of glucose uptake; C) glucose level. Zebrafish larvae were exposed to d-glucose solution (130 mM) for 72 h starting 5 dpf to induce IR, followed by treatment with GCSW210 (250 μg/mL) for 24 h before determining glucose uptake. IN 100 nM was used as positive control. Data are presented as mean ± SE (n = 8) and analyzed by one-way analysis of variance. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs control; #p < 0.05, ##p < 0.01, ###p < 0.001 vs high glucose (HG) group. Abbreviations: IN; insulin, IR; insulin resistance.

Fig. 4

Effect of GCSW210 on gene expressions in HG-mediated insulin resistance model of zebrafish larvae. A) insa; B) irs1; C) akt; D) ampka. Zebrafish larvae were exposed to d-glucose solution (130 mM) for 72 h starting 5 dpf to induce IR, followed by treatment with GCSW210 (250 μg/mL) for 24 h and homogenization. Data are presented as mean ± SE (n = 3) and analyzed by one-way analysis of variance. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus control; #p < 0.05, ##p < 0.01, ###p < 0.001 versus HG. Abbreviations: GC; subcritical-water extract of Gracilaria chorda at 210 °C (GCSW210), HG; High glucose.

Fig. 5

Effect of GCSW210 on cell cytotoxicity and glucose uptake in DEX-mediated insulin resistance model of L6 myotubes. A) cytotoxicity produced by DEX; B) glucose uptake measured at various concentrations of DEX; C) cytotoxicity after GCSW210 treatment in DEX-mediated IR model; D) glucose uptake after GCSW210 treatment in DEX-mediated IR model. L6 cells were induced to differentiate with 2% HS and treated as indicated. After 24 h of treatment with 0.5 μM of DEX, L6 myotubes were further treated with 125 or 250 μg/mL of GCSW210, or with no GCSW210 added, for 24 h. Cell viability was determined using WST assay at 450 nm and glucose uptake was measured using 2-NDBG. Data are presented as mean ± SE (n = 6) and analyzed by one-way analysis of variance followed by Dunnett test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. control; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. DEX. Abbreviations: DEX; dexamethasone, HS; horse serum, 2-NBDG; 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxyglucose.

Fig. 6

Effect of GCSW210 on to glucose metabolism-related protein expression in DEX-indused insulin resistance model of L6 myotubes. L6 myoblast were induced to differentiate with 2% HS and treated as indicated. After 24 h of treatment with 0.5 mM of DEX, L6 myotubes were further treated with 250 μg/mL of GCSW210 or with no GCSW210, for 24 h. Data are presented as mean ± SE (n = 3) and analyzed by one-way analysis of variance followed by Dunnett test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. control; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. DEX. Abbreviations: DEX; dexamethasone, GC; subcritical-water extract of Gracilaria chorda at 210 °C (GCSW210).

Fig. 7

Effect of GCSW210 on translocation of glucose transporters in DEX-induced insulin resistance model of L6 myotubes. A) cytosolic protein; B) membrane protein. Data are presented as mean ± SE (n = 3) and analyzed by one-way analysis of variance followed by Dunnett test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. control; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. DEX. Abbreviations: DEX; dexamethasone, GC; subcritical-water extract of Gracilaria chorda at 210 °C (GCSW210), GLUT4; Glucose transporter type 4.

Fig. 8

Effect of GCSW210 on PTEN protein expression in DEX-induced insulin resistance model of L6 myotubes. Data are presented as mean ± SE (n = 3) and analyzed by one-way analysis of variance followed by Dunnett test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. control; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. DEX. Abbreviations: DEX; dexamethasone, GC; subcritical-water extract of Gracilaria chorda at 210 °C (GCSW210), PTEN; phosphatase and tensin homolog.

Acknowledgments
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