Transient Silencing of a Type IV P-Type ATPase, Atp10c, Results in Decreased Glucose Uptake in C2C12 Myotubes

Abstract

Atp10c is a strong candidate gene for diet-induced obesity and type 2 diabetes. To identify molecular and cellular targets of ATP10C, Atp10c expression was altered in vitro in C2C12 skeletal muscle myotubes by transient transfection with an Atp10c-specific siRNA. Glucose uptake assays revealed that insulin stimulation caused a significant 2.54-fold decrease in 2-deoxyglucose uptake in transfected cells coupled with a significant upregulation of native mitogen-activated protein kinases (MAPKs), p38, and p44/42. Additionally, glucose transporter-1 (GLUT1) was significantly upregulated; no changes in glucose transporter-4 (GLUT4) expression were observed. The involvement of MAPKs was confirmed using the specific inhibitor SB203580, which downregulated the expression of native and phosphorylated MAPK proteins in transfected cells without any changes in insulin-stimulated glucose uptake. Results indicate that Atp10c regulates glucose metabolism, at least in part via the MAPK pathway, and, thus, plays a significant role in the development of insulin resistance and type 2 diabetes.

Figure 1

C2C12 cells were differentiated from myoblasts to myotubes as described in Section 2. Myotubes were transfected at each concentration of siRNA (SI00906220) (0, 50, 100, and 200 nM) collected at the above time points (24, 48, and 72 h). Gapdh (housekeeping gene) and Atp10c gene mRNA expression was analyzed using quantitative PCR. The percentage of knockdown was calculated at each concentration and time point based on the expression of the mock-transfected (0 nM) samples (*P < 0.7  based on Qiagen recommendations). Data represents three independent experiments with each sample repeated in triplicate.

Figure 2

C2C12 cells were differentiated from myoblasts to myotubes as described in Section 2. Myotubes were transfected at each concentration of siRNA (SI00906220) (0 and 50 nM) at the designated time point (24 h). Cells were then stimulated with insulin (100 nM, 30 min), and a 2-DOG uptake was performed (*P < 0.05). Data is reported as the glucose uptake stimulation which is expressed as the ratio of dpm/μg of total protein in presence of insulin to that in the absence of insulin.

Figure 3

(a–c): C2C12 cells were differentiated from myoblasts to myotubes as described in Section 2. Myotubes were transfected at each concentration of siRNA (SI00906220) (0 and 50 nM) and collected at the designated time point (24 h). Proteins were collected from these samples and subjected to immunoblot analysis. Data shown are representative of multiple independent experiments (n = 2 to 4), all analyzed in triplicate (*P < 0.05, ^# P < 0.1).

Figure 4

C2C12 cells were seeded onto chamber slides and allowed to differentiate from myoblasts to myotubes as described in the Materials and Methods section. Myotubes were then transfected at each concentration of siRNA (SI00906220) (0 and 50 nM) and collected at the designated time point (24 h). After transfections, cells underwent standard immunofluorescence processing and were imaged. Each sample was compared to negative and positive controls, which were used to quantify the image results (*P < 0.05).

Figure 5

C2C12 cells were differentiated from myoblasts to myotubes as described in the Materials and Methods section. Myotubes were transfected at each concentration of siRNA (SI00906220) (0 and 50 nM) at the designated time point (24 h). Cells were then treated with the inhibitor SB203580 (10 nM) and collected after 60 min. Proteins were collected from these samples and subjected to immunoblot analysis (*P < 0.05, ^# P < 0.1).

Figure 6

C2C12 cells were differentiated from myoblasts to myotubes as described in the Materials and Methods section. Myotubes were treated with the inhibitor SB203580 (10 nM) and collected after 60 min. Cells were then stimulated with insulin (100 nM, 30 min), and a 2-DOG uptake was performed (*P < 0.05, ^# P < 0.1).