Fig 1.
Analysis of p-Akt in mouse and human skeletal muscle cells treated with insulin, zinc, and NaPy over 60 min.
Top and bottom panel represents mouse and human skeletal muscle cells, respectively. Time is shown in minutes from 0, 15, 30 and 60 and total Akt was used as an internal loading control in both panels and levels of pAKT were normalized to total Akt.
Fig 2.
Analysis of p-ERK in mouse and human skeletal muscle cells treated with insulin, zinc, and NaPy over 60 min.
Top and bottom panel represents mouse and human skeletal muscle cells, respectively. Time is shown in minutes from 0, 15, 30 and 60 and total ERK was used as an internal loading control in both panels and levels of p-ERK were normalized to total ERK.
Fig 3.
Analysis of p-SHP in mouse and human skeletal muscle cells treated with insulin, zinc, and NaPy over 60 min.
Top and bottom panel represents mouse and human skeletal muscle cells, respectively. Time is shown in minutes from 0, 15, 30 and 60 and total SHP was used as an internal loading control in both panels and levels of p-SHP were normalized to total SHP.
Fig 4.
Analysis of p-tyrosine in mouse and human skeletal muscle cells treated with insulin, ZnSO4, and NaPy over 60 min.
Top and bottom panel represents mouse and human skeletal muscle cells, respectively. Time is shown in minutes from 0, 15, 30 and 60 and GAPDH was used as an internal loading control in both panels and levels of p-tyrosine were normalized to GAPDH.
Fig 5.
Effect of insulin and zinc on intracellular proteins via analysis of an intracellular protein array (see Table 1).
a. Mouse skeletal muscle cells. Top panel, control (untreated cells), middle panel, insulin treated cells; bottom panel, ZnSO4 treated cells. Positive and negative controls are shown labelled on the control panel. Significant responses to insulin and ZnSO4 (PRAS40, Akt, ERK1/2 and GSK-3β) are shown. b. Human skeletal muscle cells. Top panel, control (untreated cells), middle panel, insulin treated cells; bottom panel, ZnSO4 treated cells. Positive and negative controls are shown labelled on the control panel. Significant responses to insulin (PRSA40, Akt and ERK1/2) and ZnSO4 (Akt, ERK1/2, GSK-3β and p38) are shown. All positive signaling molecules in the insulin and zinc treated arrays were normalized to their corresponding signaling molecule on the control array (no treatment).
Table 1.
Protein target map of intracellular signaling array (cell signaling).
Fig 6.
Densitometry results from Fig 5 protein array in mouse skeletal muscle cells.
a. pAkt, b. pERK1/2, c. GSK-3β, and d. PRAS40. Control (untreated cells), ZnSO4 and insulin treatments are shown. Differences between the expressions of proteins in cells were determined by one-way ANOVA. The results of 3 independent experiments are presented as a mean ± standard error. *p < 0.05, ** p < 0.01 and *** p <0.001 considered significant when compared to the control.
Fig 7.
Densitometry results from Fig 5 protein array in human skeletal muscle cells.
a. pAkt, b. pERK1/2, c. pGSK-3β, d. PRAS40, and e. p38. Control (untreated cells), ZnSO4 and insulin treatments are shown. Differences between the expressions of proteins in cells were determined by one-way ANOVA. The results of 3 independent experiments are presented as a mean ± standard error. * p < 0.05, ** p < 0.01 and *** p <0.001 considered significant when compared to the control.
Fig 8.
Glucose oxidation assay in the presence of insulin and ZnSO4.
a. Mouse skeletal muscle cells, and b. Human skeletal muscle cells. The results of 3 independent experiments are presented as a mean ± standard error. * p < 0.05, ** p < 0.01 and *** p <0.001 considered significant compared with the control (untreated cells).
Fig 9.
Effect of insulin receptor tyrosine kinase inhibitor HNMPA-(AM)3 on insulin and zinc-induced pAkt.
Mouse C2C12 skeletal muscle cells were treated with increasing concentrations (0, 25, 50, and 100 μM) of HNMPA-(AM)3 followed by either treatment with insulin (10 nM) or zinc (20 μM) for 1 hour. Concentrations of HNMPA-(AM)3 are given at 0, 25, 50 and 100 μM. Top panel: insulin-treated measurement of pAkt, Lower panel: zinc-treated measurement of pAkt. Total Akt was used as an internal loading control in both panels and levels of pAKT were normalized to total Akt.