Autophagy induction in liver, skeletal muscle and brain tissues . 48

starvation-induced autophagy indicates mTORC1 inactivation (Kim et al. 2011), it was examined to monitor mTORC1 activity in different mouse tissues. The lysosome-mediated degradation of cytosolic material could be enhanced as a response to cellular damage and nutrient deprivation (Reviewed by Singh and Cuervo 2011;

Reviewed by Yim and Mizushima 2020). Microtubule-associated protein 1 light chain 3 (LC3) is an indicator of autophagosome biogenesis via its conversion of a cytosolic LC3-I form to lipid bound LC3-II form, which binds to the autophagic membranes (Kabeya et al. 2000). LC3 has three isoforms, namely LC3A, B and C, with the first two being preserved in mice (Cann et al. 2008).

In this experiment, LC3B isoform was used as an autophagy marker. In the presence and absence of food intake, autophagy activation was monitored in liver, skeletal muscle and brain tissues via western blot. SDS-PAGE separated proteins were blotted and probed with LC3B and GAPDH antibodies. Since LC3B-II is bound to the autophagosomal membrane and is subject to lysosomal degradation, the detected signals of LC3B-II normalized to GAPDH correspond to the levels of autophagosomes (Reviewed by Klionsky et al. 2016). For a more informative picture of the cellular autophagic response, LC3B-II was normalized to LC3B-I and the ratio was measured (Kabeya et al. 2004; Tanida et al. 2005).

At each starvation condition, three biological replicates were used. The distribution of the sample means was measured via the standard error of the mean (SEM) after setting the control samples to 1.

Figure 4.8 A shows the detected signals of the 16 kDa LC3B-I and the 14 kDa LC3B-II as well as GAPDH as a loading control in liver tissues. The GAPDH-normalized LC3B-II intensities depicted a gradual increase reaching their highest after 9 and 12 hours of starvation with a statistical significant increase of 3.68- and 3.64-fold, respectively, compared to control samples. A 1.82-fold increase

was also measured between the control and 16 hours starved samples.

Subsequently, the intensities decreased by 2.02- and 2.84-fold after 16 and 20 hours compared to 9 hours of starvation, respectively. In addition, a decrease of 2.81-fold was monitored between 12 and 20 hours of starvation. In parallel, the measured LC3B-II/LC3B-I ratio in liver lysates showed a similar pattern with an increase of 1.51- and 1.58-fold after 9 and 12 hours of starvation, respectively, compared to control samples. The ratio then decreased by 2.6- and 2.73-fold after 20 hours compared to 9 and 12 hours, respectively.

Figure 4.8: Levels of LC3B in different tissues of starved mice

Adult C57BL/6 mice were starved for 6, 9, 12, 16 or 20 hours and control mice were fed ad libitum before sacrifice. After liver, skeletal muscle and brain 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 LC3B as an autophagosome marker and GAPDH as a loading control. A-C. The protein abundance was densitometrically quantified and LC3B-II signals were normalized to GAPDH and LC3B-I intensities. The average relative intensities are shown as bar charts for the three different tissues. 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.

Results 50

In skeletal muscle, figure 4.8 B displays the protein amounts of LC3B-I and -II together with GAPDH. The intensities of normalized LC3B-II demonstrated no statistical significant change throughout the starvation course. Meanwhile, the abundance of LC3B-II signals was getting more distinct than LC3B-I the longer the starvation occurs. This shift was also measured after applying the unpaired student’s t-test to the LC3B-II/LC3B-I ratio. At the early starvation time periods, no change in the intensities was monitored. Next, after 16 hours, LC3B-II/LC3B-I ratio increased by 9.21-, 6.66-, 8.46- and 3.12-fold compared to control, 6, 9 and 12 hours starved samples, respectively.

In figure 4.8 C, brain homogenates were incubated with LC3B and GAPDH specific antibodies and a very low abundance of LC3B-II was observed. LC3B-II signals were normalized to GAPDH and no statistical significant change was measured except for a 1.14-fold decrease between 6 and 12 hours of starvation.

At the same time, LC3B-II signals were normalized to LC3B-I and the intensities decreased by 1.41- and 1.2-fold after 16 and 20 hours, respectively, compared to 6 hours of starvation. No further significant change was recorded.

These findings suggest that autophagy takes place when mTORC1 is least active, consistent with previous observations of protein synthesis regulation by mTORC1.

4.2 Analysis of short- and long-term starvation and their impact on body metabolism in wild-type mice

Metabolism of the main energy sources glucose, fatty acids and amino acids can vary as a response to different starvation situations. Although starvation is usually accompanied by weight and energy loss, the body adapts by producing other fuel molecules such as ketone bodies (Berg et al. 2002a).

Findings of starvation experiments in key metabolic organs demonstrated interesting response mechanisms to short and prolonged nutrient deprivation on the molecular level. In liver tissues, phosphorylation of downstream targets by mTORC1 was decreased after short starvation and autophagy occurred. In contrast, prolonged starvation demonstrated a reactivation in downstream targets phosphorylation and a decline in starvation-mediated autophagy. Therefore, two

starvation periods with different molecular impacts, namely 6 and 24 hours, were selected and the effect of starvation on body and cellular metabolism was investigated.

From control, 6 and 24 hours starved mice for later lysosome enrichment experiment, the body weight and the whole blood glucose levels were tested too.

In addition, the concentrations of ketone bodies, acylcarnitines, amino acids and their derivatives were also measured in the serum of the same sacrificed mice.