Methyl Syringate Stimulates Glucose Uptake by Inhibiting Protein Tyrosine Phosphatases Relevant to Insulin Resistance

Abstract

Several protein tyrosine phosphatases (PTPs), particularly PTPN1, PTPN2, PTPN6, PTPN9, PTPN11, PTPRS, and DUSP9, are involved in insulin resistance. Therefore, these PTPs could be promising targets for the treatment of type 2 diabetes. Our previous studies revealed that PTPN2 and PTPN6 are potential antidiabetic targets. Therefore, the identification of dual-targeting inhibitors of PTPN2 and PTPN6 could be a potential therapeutic strategy for the treatment or prevention of type 2 diabetes. In this study, we demonstrate that methyl syringate inhibits the catalytic activity of PTPN2 and PTPN6 in vitro, indicating that methyl syringate acts as a dual-targeting inhibitor of PTPN2 and PTPN6. Furthermore, methyl syringate treatment significantly increased glucose uptake in mature 3T3-L1 adipocytes. Additionally, methyl syringate markedly enhanced phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) in 3T3L1 adipocytes. Taken together, our results suggest that methyl syringate, a dual-targeting inhibitor of PTPN2 and PTPN6, is a promising therapeutic candidate for the treatment or prevention of type 2 diabetes.

Keywords: PTPN2; PTPN6; catalytic activity; glucose uptake; methyl syringate; protein tyrosine phosphatases (PTPs); type 2 diabetes.

Figure 1

Suppression of PTPN2 and PTPN6 enhances phosphorylation. (A–C) 3T3-L1 preadipocytes were transfected with PTPN2, PTPN6 siRNAs, or scrambled siRNA (control siRNA). After 48 h, cells were lysed and analyzed using Western blotting (A,B) or quantitative real-time PCR (C). (B) Quantification of phospho-AMPK and total-AMPK using ATTO image analysis software. Results are expressed as mean ± standard error of the mean (SEM). Data were analyzed using a two-tailed unpaired t-test. *** p < 0.001; ** p < 0.01 compared to the control siRNA.

Figure 2

Methyl syringate inhibits the catalytic activity of PTPN2 and PTPN6. (A,B) PAGE analysis of PTPN2 ((A) molecular weight: 89.7 kDa) and PTPN6 ((B) molecular weight: 67.6 kDa). M, molecular weight marker; lane 1, total sample before induction; lane 2, supernatant before induction; lane 3, total sample after induction; lane 4, supernatant after induction; lane 5, sample passed through the column after sonication; lane 6, sample after washing the column using lysis buffer; lane 7, sample after washing the column using 10 mM imidazole’ lanes 8 and 9, protein eluted using 100 mM imidazole. (C,D) IC₅₀ values for methyl syringate were estimated using a sigmoid curve against percent inhibition (%) for log[Methyl syringate] (μM). (E,F) The Hill coefficient (n_H) was obtained from slopes of Hill plots using the Hill equation.

Figure 3

Methyl syringate enhances glucose uptake. (A) Differentiated 3T3-L1 adipocytes were incubated with low-glucose DMEM for 16 h. Subsequently, the cells were then treated with 5, 10, or 20 μM methyl syringate in glucose-depleted DMEM for 6 h, and cell viability was assessed. (B) 3T3-L1 preadipocytes were differentiated in the presence of 5, 10, or 20 μM methyl syringate for 3 days, and cell viability was evaluated. (C,D) Differentiated 3T3-L1 adipocytes (C) and C2C12 muscle cells (D) were incubated with 10 and 20 μM methyl syringate, control (0.1% dimethyl sulfoxide treatment group), or 0.1 μM insulin (positive control) for 6 h (methyl syringate and control) or 30 min (insulin). Next, the cells were maintained with the fluorescent glucose indicator 2-NBDG for 1 h, and the fluorescence intensity was measured. Results are presented as mean ± SEM. Data were analyzed using a two-tailed unpaired t-test. **** p < 0.0001; *** p < 0.001 compared to the control group.

Figure 4

Methyl syringate increases AMPK phosphorylation. (A) 3T3-L1 adipocytes were treated with of 5, 10, and 20 μM methyl syringate for 6 h, and Western blotting was carried out using antibodies against phosphorylated AMPK, total AMPK, and β-actin. (B) Quantification of phospho-AMPK/total-AMPK using the ATTO image analysis software. Results are presented as mean ± SEM. Data were analyzed using a two-tailed unpaired t-test. *** p < 0.001; ** p < 0.01 compared to the control group.

Figure 5

Methyl syringate does not enhance lipid accumulation. (A) 3T3-L1 preadipocytes were differentiated into mature adipocytes in the presence of 10 or 20 μM methyl syringate. Lipid droplets was evaluated by Oil Red O staining on day 6 after differentiation. (B) To quantify lipid accumulation, Oil Red O dye was eluted by treatment with isopropanol, and the absorbance was measured at 490 nm using a microplate reader. Results are presented as mean ± SEM. Scale bars: 100 μm.