The regulation of skeletal muscle protein turnover during the progression of cancer cachexia in the Apc(Min/+) mouse

Abstract

Muscle wasting that occurs with cancer cachexia is caused by an imbalance in the rates of muscle protein synthesis and degradation. The Apc(Min/+) mouse is a model of colorectal cancer that develops cachexia that is dependent on circulating IL-6. However, the IL-6 regulation of muscle protein turnover during the initiation and progression of cachexia in the Apc(Min/+) mouse is not known. Cachexia progression was studied in Apc(Min/+) mice that were either weight stable (WS) or had initial (≤5%), intermediate (6-19%), or extreme (≥20%) body weight loss. The initiation of cachexia reduced %MPS 19% and a further ∼50% with additional weight loss. Muscle IGF-1 mRNA expression and mTOR targets were suppressed with the progression of body weight loss, while muscle AMPK phosphorylation (Thr 172), AMPK activity, and raptor phosphorylation (Ser 792) were not increased with the initiation of weight loss, but were induced as cachexia progressed. ATP dependent protein degradation increased during the initiation and progression of cachexia. However, ATP independent protein degradation was not increased until cachexia had progressed beyond the initial phase. IL-6 receptor antibody administration prevented body weight loss and suppressed muscle protein degradation, without any effect on muscle %MPS or IGF-1 associated signaling. In summary, the %MPS reduction during the initiation of cachexia is associated with IGF-1/mTOR signaling repression, while muscle AMPK activation and activation of ATP independent protein degradation occur later in the progression of cachexia. IL-6 receptor antibody treatment blocked cachexia progression through the suppression of muscle protein degradation, while not rescuing the suppression of muscle protein synthesis. Attenuation of IL-6 signaling was effective in blocking the progression of cachexia, but not sufficient to reverse the process.

Figure 1. Muscle protein synthesis and IGF-1/mTOR signaling are reduced during the progression of cachexia in Apc^Min/+ mice.

Protein synthesis and IGF-1 expression were measured in Apc^Min/+ mice during the progression of cachexia. A) Myofibrillar protein synthesis. B) Correlation between muscle weights and protein synthesis. C) Upper: representative western blot of phosphorylated and total forms of Akt (Ser 473), mTOR (Ser2448), p70S6k (Thr389) and 4EBP-1 (Thr37/46). Lower: The ratio of phosphorylated and total Akt, mTOR, p70 and 4EBP1 in the gastrocnemius muscle normalized to the WS group. D) IGF-1 expression and E) correlation between IGF-1 gene expression and protein synthesis. F) Skeletal alpha actin mRNA expression. Values are means ± SE. Significance was set at p<0.05. † Signifies different from WS mice. & Signifies difference from mice with ≤5% body weight loss. $ Signifies difference from mice with 6–19% body weight loss. WS, weight stable.

Figure 2. AMPK activation increases during late stage cachexia.

AMPK activation was measured in Apc^Min/+ mice during the progression of cachexia. A) AMPK activity in the gastrocnemius muscle normalized to the WS mice. B) Upper: representative western blot of phosphorylated AMPK (Thr172) and total AMPK in the gastrocnemius. Lower: The ratio of phosphorylated to total forms of AMPK in the gastrocnemius muscle. C) Upper: representative western blot of phosphorylated raptor (Ser792) and total raptor in the gastrocnemius. Lower: The ratio of phosphorylated to total forms of raptor in the gastrocnemius muscle. Values are means ± SE. Significance was set at p<0.05. † Signifies different from WS mice. & Signifies difference from mice with ≤5% body weight loss. $ Signifies difference from mice with 6–19% body weight loss. WS, weight stable.

Figure 3. Skeletal muscle protein degradation consists of ATP dependent and independent mechanisms during the progression of cachexia in the Apc^Min/ mice.

Protein degradation was measured in Apc^Min/+ mice with progressive body weight loss. A) Protein degradation determined by tyrosine release assay. White bars represent ATP dependent degradation; Black bars represent ATP independent degradation. B) Correlation between protein degradation and gastrocnemius weight. C) Correlation between protein degradation and the percentage of body weight loss. D) Upper representative western blot of ubiquitinated proteins in the gastrocnemius muscle. Lower Quantification of ubiquitinated proteins normalized to weight stable Apc^Min/+ mice. E) Gene expression of C7 and C2 proteasomal subunits. F). Upper: representative western blot of phosphorylated and total forms of Foxo3. Lower: The ratio of phosphorylated and total Foxo3. G) Gene expression of atrogin1 and MuRF1. H) Representative western blot of atrogin1 protein. Values are means ± SE. Significance was set at p<0.05. † Signifies different from WS groups. & Signifies difference from mice with ≤5% body weight loss. WS, weight stable.

Figure 4. Autophagy is increased during late stage cachexia in the Apc^Min/+ mouse.

A) Upper representative western blot of Beclin-1 protein. Lower Quantification of Beclin-1 protein normalized to weight stable Apc^Min/+ mice, B) Upper representative western blot of Atg7 protein. Lower Quantification of Atg7 protein. C) Upper representative western blot of LC3β protein. Lower Quantification of LC3β protein. Values are means ± SE. Significance was set at p<0.05. † Signifies difference from weight stable Apc^Min/+ mice. $ Signifies different from Apc^Min/+ mice with 6–19% body weight loss. WS, weight stable.

Figure 5. Inhibition of IL-6 signaling attenuates the progression of cachexia independent of changes in protein synthesis.

Wild type and Apc^Min/+ mice were given an IL-6 receptor antibody or PBS control at 16 weeks of age and sacrificed at 18 weeks. A) Experimental design showing administration schedule and time line of IL-6 receptor antibody or PBS treatment. B) Percentage body weight change from peak body weight to sacrifice weight. C) Polyp distribution presented in percentage of total tumors. D) Representative western blot of phosphorylated and total forms of Stat3 and total SOC3 protein expression. E) Gastrocnemius weight and F) muscle protein synthesis measured at sacrifice. G) IGF-1 gene expression in the gastrocnemius muscle. H) Upper: Representative western blots of phosphorylated and total forms of Akt, mTOR, p70S6K, and 4E-BP1. Lower: The ratio of phosphorylated to total form of the given protein normalized to PBS treated wild-type mice. Values are means ± SE. Significance was set at p<0.05. *Signifies difference within treatment. † Signifies difference within genotype. # Signifies main effect of genotype. & Signifies difference in polyps 1–2 mm. $ Signifies difference between polyps >2 mm.

Figure 6. Administration of IL-6 receptor antibody attenuates the increase of phosphorylation of AMPK and raptor.

A). Upper: representative western blot of phosphorylated and total forms of AMPK. Lower: The ratio of phosphorylated and total AMPK expression normalized to wild-type PBS control. B). Upper: representative western blot of phosphorylated and total forms of raptor. Lower: The ratio of phosphorylated and total forms of raptor. Values are means ± SE. Significance was set at p<0.05. *Signifies difference within treatment. † Signifies difference within genotype.

Figure 7. IL-6 inhibition by IL-6 receptor antibody attenuates protein degradation and ubiquitin proteasomal degradation.

Wild type and Apc^Min/+ mice were given an IL-6 receptor antibody or PBS control at 16 weeks of age and sacrificed at 18 weeks. A) Protein degradation determined by tyrosine release assay. White bars represent ATP dependent degradation; Black bars represent ATP independent degradation. B) Upper representative western blot of ubiquitinated proteins in the gastrocnemius muscle. Lower Quantification of ubiquitinated proteins normalized to wild-type PBS control mice. C) Gene expression of C7 and C2 proteasomal subunits. D) Upper: representative western blot of phosphorylated and total forms of Foxo3. Lower: The ratio of phosphorylated and total Foxo3. E) Gene expression of atrogin1 and MuRF1. F) Representative western blot of atrogin1 protein. Values are means ± SE. Significance was set at p<0.05. *Signifies difference from wild-type control. † Signifies difference within Apc^Min/+ mice.

Figure 8. The induction of autophagy is attenuated by IL-6 inhibition.

A) Upper representative western blot of Beclin-1 protein. Lower Quantification of Beclin-1 protein normalized to wild-type PBS control mice. B) Upper representative western blot of Atg7 protein. Lower Quantification of Atg7 protein normalized to wild-type PBS control mice. C) Upper representative western blot of LC3β protein. Lower Quantification of LC3β protein normalized to wild-type PBS control mice. Values are means ± SE. Significance was set at p<0.05. *Signifies difference from wild type control. † Signifies difference within Apc^Min/+ mice.