Induction of in vitro ketosis condition and suppression using methylsulfonylmethane by altering ANGPTL3 expression through STAT5b signaling mechanism

Figures & data

Figure 1. MSM inhibited the viability of mouse hepatocyte, FL83B cells in a dose dependent manner.

Note: MTT assay for cell proliferation analysis by MSM in FL83B cells. A total of 30 or 40 mM MSM used for further experiments.

Figure 2. Ketosis cell model construction and inhibition of glucose starvation induced ketosis by MSM in FL83B cells.

Note: Analysis of ketone body formation using BHB colorimetric assay: (a) BHB standard graph. (b) Estimation of BHB concentration (mM) after glucose starvation (3%), treatment with high glucose (9%), low glucose plus 30 or 40 mM MSM for 24 h. (c) Relative expression (%) of BHB with respect to the low glucose ketosis group. Statistical analysis was done by using ANOVA test. L Glc, low glucose (3%); H Glc, high glucose (9%); *P < 0.05 and **P < 0.001.

Figure 3. MSM upregulated the expression of GHR in ketosis condition.

Note: (a) Western blotting analysis of GHR expression in whole cell lysate after treatment with high glucose, low glucose plus 30 or 40 mM MSM for 24 h. (b) Relative expression of GHR with respect to β-actin. Statistical analysis was done by using ANOVA test. L Glc, low glucose (3%); H Glc, high glucose (9%); **P < 0.001.

Figure 4. MSM downregulated the expression of ANGPTL3 in ketosis condition.

Note: (a) RT-PCR analysis of ANGPTL3 after the treatment with high glucose, low glucose plus 30 or 40 mM MSM for 24 h. (b) Relative decreases of ANGPTL3 mRNA level by high glucose, low glucose plus 30 or 40 mM MSM with respect to 18s RNA. (c) Western blotting analysis showing protein level inhibition of ANGPTL3 by high glucose, low glucose plus 30 or 40 mM MSM. (d) Relative decreases of ANGPTL3 protein level by high glucose, low glucose plus 30 or 40 mM MSM in whole cell lysates. Statistical analysis was done by using ANOVA test. L Glc, low glucose (3%); H Glc, high glucose (9%); *P < 0.001.

Figure 5. MSM enhanced STAT5b/IGF-1R signaling and STAT5b/DNA binding activity.

Note: (a) Western blotting analysis of whole cell lysate after treatment with high glucose, low glucose plus 30 or 40 mM MSM for 24 h. (b) Nuclear protein analysis after the treatment with high glucose, low glucose plus 30 or 40 mM MSM for 24h by western blotting. (c) DNA binding activities of STAT5b to the GAS element was upregulated by the MSM, analyzed by gel shift assay. Data shown are one representative of three independent experiments.

Figure 6. STAT5b is a negative regulator for ANGPTL3 in ketosis condition.

Note: (a) On-target inhibition of STAT5b reversed the expression pattern of ANGPTL3 after silenced STAT5b. (b) Graphical representation of the relative expression of ANGPTL3 with respect to β-actin. Statistical analysis was done by using Student’s t-test; ***p < 0.001.