Gracilaria chorda subcritical-water extracts as ameliorant of insulin resistance induced by high-glucose in zebrafish and dexamethasone in L6 myotubes

Abstract

Background and aim: Insulin resistance (IR) is a pathological condition in which cells fail to respond normally to insulin. Loss of insulin sensitivity disrupts glucose homeostasis and elevates the risk of developing the metabolic syndrome that includes Type 2 diabetes. This study assesses the effect on subcritical-water extract of Gracilaria chorda (GC) at 210 °C (GCSW210) in IR induction models of high glucose (HG)-induced zebrafish larvae and dexamethasone (DEX)-induced L6 myotubes.

Experimental procedure: The dose of HG and DEX for IR induction in zebrafish larvae and L6 myotubes was 130 mM or 0.5 μM. The capacity of glucose uptake was quantified by fluorescence staining or intensity. In addition, the activation of protein and mRNA expressions for insulin signaling (insulin-dependent or independent pathways) was measured.

Results and conclusion: Exposure of zebrafish larvae to HG significantly reduced the intracellular glucose uptake with dose-dependnet manner compared to control. However, the group treated with GCSW210 significantly averted HG levels like the insulin-treated group, and significantly up- or down-regulated the mRNA expressions related to insulin production (insα) and insulin signaling pathways. Moreover, the treatment with GCSW210 effectively regulated the protein expression of PI3K/AKT, AMPK, and GLUT4 involved in the action of insulin in IR models of L6 myotubes compared to DEX-treated control. Our data indicate that GCSW210 stimulates activation of PI3K/AKT and AMPK pathways to attenuate the development of IR induced by HG in zebrafish and DEX in L6 myotubes. In conclusion, GCSW210 is a potential agent for alleviating various diseases associated with the insulin resistance.

Keywords: Gracilaria chorda; Insulin resistance; L6 myotubes; Subcritical-water extract; Zebrafish larvae.

Graphical abstract

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.