Effect of starvation on mTORC1 activity in skeletal muscle

(Baskin et al. 2015). Based on this fact and after showing mTORC1 activation pattern in the liver, the influence of starvation on mTORC1 in starved skeletal muscle tissues was examined. The investigation was performed in three biological replicates from control and starved mice by western blot analysis. For signal quantification, obtained intensities were normalized to GAPDH signals as a loading control. Obtained signals at 15-20 kDa and 37 kDa representing p-4EBP1 and GAPDH, respectively, are visualized in figure 4.4 A. Figure 4.4 B represents the normalized results of p-4EBP1 at each starvation period after setting the control sample to 1, with a decrease in the intensities appearing from the early starvation periods. The significance of the normalized intensities was measured using the unpaired student’s t-test. By comparing the control sample to the starvation time periods, there is a gradual decrease of signal intensity until 12 hours of starvation. After 16 and 20 hours the signal increases slightly, but stays significantly below the control values. Compared to the fed state, 4EBP1 phosphorylation decreased by 1.60-, 7.85-, 4.58- and 2.41-fold after 9, 12, 16 and 20 hours of starvation, respectively. Compared to 9 hours starved samples, a 4.89- and 2.86-fold decrease was measured after 12 and 16 hours of starvation, respectively. In figure 4.4 C, samples were subjected to western blotting using antibodies directed against total 4EBP1 and GAPDH proteins. Figure 4.4 D depicts the quantification of immune detected signals relative to control samples which were set to 1. The amount of gamma isoform, the uppermost phosphorylated isoform, dropped starting from 12 hours in contrast to increased alpha isoform amount, showing a reduction in 4EBP1 phosphorylation. In parallel, the beta isoform levels were not affected by starvation. Statistical analysis was applied to gamma, beta and alpha isoforms as well as the sum of all isoforms at each starvation period, and demonstrated no statistical change in the intensities at the early starvation time periods. On average, the gamma isoform revealed a significant decrease of 3.4-, 2.78- and 2.73-fold compared to control, 6 and 9 hours, respectively, starting from 12 hours of starvation. In contrast, alpha isoform amounts increased substantially after 12 and 16 hours compared to control, 6

and 9 hours starved samples. The increase was by 3.13- and 2.76-fold (control), 2.62- and 2.33-fold (6 hours) as well as 3.1- and 2.75-fold (9 hours), all after 12 and 16 hours, respectively.

Figure 4.4: Investigation of mTORC1 activity in skeletal muscle tissues by 4EBP1 immune detection

Adult C57BL/6 mice were starved for 6, 9, 12, 16 or 20 hours and control mice were fed ad libitum before sacrifice. After skeletal muscle tissues were removed and lysed, 12 µg proteins were separated on a 12.5 % SDS-PAGE gel, transferred onto a nitrocellulose membrane and probed with antibodies. A. Obtained signals from p-4EBP1 (T37/46) and the loading control GAPDH were visualized. B. Densitometric quantified and normalized intensities are depicted as bar charts. C. Western blot signals of total 4EBP1 and GAPDH proteins were displayed. D. For all conditions and isoforms, intensities were normalized to GAPDH signals. Shown are mean + SEM; n=3 and the significance was determined by unpaired student’s t-test (*=p<0.05; **=p<0.01). The control samples were set to 1.

The comparison of the sum of the isoforms under all conditions depicted no significant change except for an increase of 1.22-fold between the control and 16 hours starved samples. In addition, a 1.32- and 1.4-fold increase after 12 and 16 hours was calculated relative to 9 hours. As a measure of the mean distribution

Results 44

of the samples, the standard error of the mean (SEM) was applied (Figure 4.4 B, D).

In addition to 4EBP1, signals of total S6 and p-S6 were detected by western blot and GAPDH-normalized intensities were subjected to the unpaired student’s t-test. Figure 4.5 A shows the immune detected signals of p-S6 and the loading control GAPDH where low protein abundances were seen in all starvation periods relative to control samples except of 9 hours starved samples.

Figure 4.5: Investigation of mTORC1 activity in skeletal muscle tissues by S6 immune detection

Adult C57BL/6 mice were starved for 6, 9, 12, 16 or 20 hours and control mice were fed ad libitum before sacrifice. After skeletal muscle tissues were removed and lysed, 12 µg proteins were separated on a 12.5 % SDS-PAGE gel, transferred onto a nitrocellulose membrane and probed with antibodies. A. Western blotting of p-S6 (S240/244) and the loading control GAPDH. B. Quantified signals were normalized to the corresponding loading control intensities. C. Total S6 and GAPDH signals were detected in skeletal muscle samples of control and starved mice. D. The level of total S6 in each sample was normalized to the GAPDH level. Shown are mean + SEM; n=3 and the significance was determined by unpaired student’s t-test (*=p<0.05). The control samples were set to 1.

After setting the control sample to 1, the average of normalized p-S6 with the SEM are depicted in figure 4.5 B, where S6 phosphorylation showed a statistical

decrease of 1.43-fold between 12 and 16 hours of starvation. Although p-S6 abundance showed a decrease up from 6 hours of starvation, however this decrease was not statistically significant. The second replicate of 9 hours starved mice, which showed a strong signal intensity, was considered an outlier and was excluded from the calculation of the mean abundance. Therefore, the unpaired student’s test is not reliable at 9-hour starvation period.

As for p-S6, total S6 together with GAPDH signals are visualized in figure 4.5 C and the relative intensities as well as the standard error of the mean are shown in figure 4.5 D. The obtained signals showed a varying pattern throughout the time course of starvation. The unpaired student’s t-test showed a statistical decrease of 3.11-, 2,41- and 1.96-fold after 16 hours in correlation with control, 6 and 12 hours starved samples, respectively.

Regarding the results obtained from the starved skeletal muscle tissues, phosphorylation of 4EBP1 and S6 indicates a gradually decreased level by increasing the duration of starvation.