IL-6 regulation on skeletal muscle mitochondrial remodeling during cancer cachexia in the ApcMin/+ mouse

Abstract

Background: Muscle protein turnover regulation during cancer cachexia is being rapidly defined, and skeletal muscle mitochondria function appears coupled to processes regulating muscle wasting. Skeletal muscle oxidative capacity and the expression of proteins regulating mitochondrial biogenesis and dynamics are disrupted in severely cachectic ApcMin/+ mice. It has not been determined if these changes occur at the onset of cachexia and are necessary for the progression of muscle wasting. Exercise and anti-cytokine therapies have proven effective in preventing cachexia development in tumor bearing mice, while their effect on mitochondrial content, biogenesis and dynamics is not well understood. The purposes of this study were to 1) determine IL-6 regulation on mitochondrial remodeling/dysfunction during the progression of cancer cachexia and 2) to determine if exercise training can attenuate mitochondrial dysfunction and the induction of proteolytic pathways during IL-6 induced cancer cachexia.

Methods: ApcMin/+ mice were examined during the progression of cachexia, after systemic interleukin (IL)-6r antibody treatment, or after IL-6 over-expression with or without exercise. Direct effects of IL-6 on mitochondrial remodeling were examined in cultured C2C12 myoblasts.

Results: Mitochondrial content was not reduced during the initial development of cachexia, while muscle PGC-1α and fusion (Mfn1, Mfn2) protein expression was repressed. With progressive weight loss mitochondrial content decreased, PGC-1α and fusion proteins were further suppressed, and fission protein (FIS1) was induced. IL-6 receptor antibody administration after the onset of cachexia improved mitochondrial content, PGC-1α, Mfn1/Mfn2 and FIS1 protein expression. IL-6 over-expression in pre-cachectic mice accelerated body weight loss and muscle wasting, without reducing mitochondrial content, while PGC-1α and Mfn1/Mfn2 protein expression was suppressed and FIS1 protein expression induced. Exercise normalized these IL-6 induced effects. C2C12 myotubes administered IL-6 had increased FIS1 protein expression, increased oxidative stress, and reduced PGC-1α gene expression without altered mitochondrial protein expression.

Conclusions: Altered expression of proteins regulating mitochondrial biogenesis and fusion are early events in the initiation of cachexia regulated by IL-6, which precede the loss of muscle mitochondrial content. Furthermore, IL-6 induced mitochondrial remodeling and proteolysis can be rescued with moderate exercise training even in the presence of high circulating IL-6 levels.

Figure 1

Mitochondrial content, biogenesis and morphology are altered during the progression of cachexia.Apc^Min/+ mice were grouped by percentage of body weight loss to study muscle oxidative capacity during the progression of cachexia. A) Mitochondrial content as determined by the mitochondrial:nuclear DNA ratio. B) Representative Western blot of cytochrome C, CoxIV and PGC-1α protein expression throughout the progression of cachexia. C) Cytochrome C, D) CoxIV and E) PGC-1α protein expression normalized to weight stable mice. Representative EM images of intramuscular mitochondria in F) wild-type, G) Apc^Min/+ mice with mild cachexia and H) Apc^Min/+ mice with severe cachexia. I) Mitochondrial size and J) mitochondrial size distribution. Values are means ± SE. Significance was set at P <0.05. † Signifies different from weight stable groups. $ Signifies difference from mice with 6 to 19% body weight loss. WS, weight stable.

Figure 2

Mitochondrial dynamics are altered during the progression of cachexia. A) Representative Western blot of Mfn1, Mfn2 and FIS1 protein expression during the progression of cachexia. B) Mfn1, C) Mfn2 and D) FIS1 protein expression normalized to weight stable mice. E) Bax mRNA expression normalized to weight stable mice. Values are means ± SE. Significance was set at P <0.05. † Signifies the difference from WS groups. & Signifies the difference from mice with ≤5% body weight loss. $ Signifies the difference from mice with 6 to 19% body weight loss. WS, weight stable.

Figure 3

IL-6 inhibition attenuates the loss in mitochondrial content and biogenesis in theApc^Min/+mouse. Wild-type and Apc^Min/+ mice were treated with an IL-6 receptor antibody for two weeks to inhibit IL-6 signaling. A) Mitochondrial content as determined by the mitochondrial:nuclear DNA ratio. B) Representative Western blot of Cytochrome C, Cox IV and PGC-1α protein in wild-type and Apc^Min/+ mice treated with an IL-6 receptor antibody or PBS control. C) Cytochrome C, D) CoxIV and E) PGC-1α protein expression normalized to wild-type PBS control mice. Values are means ± SE. Significance was set at P <0.05. *Signifies the difference within treatment. † Signifies the difference within genotype.

Figure 4

IL-6 inhibition restores mitochondrial dynamics and reduces apoptosis in theApc^Min/+mouse. A) Representative Western blot of Mfn2 and FIS1 protein expression in wild-type and Apc^Min/+ mice treated with an IL-6 receptor antibody or PBS control. B) Mfn2 and C) FIS1 protein expression normalized to PBS treated wild-type mice. D) Bax mRNA expression normalized to PBS treated wild-type mice. Values are means ± SE. Significance was set at P <0.05. * Signifies the difference within treatment. † Signifies the difference within genotype.

Figure 5

IL-6 over-expression induced muscle wasting and reduced PGC-1α which are rescued with exercise training.Apc^Min/+ mice underwent 12 weeks of moderate exercise training or served as sedentary cage controls and over-expressed circulating IL-6 or received a control vector. A) Gastrocnemius muscle mass. B) Representative Western blot of cytochrome C, CoxIV and PGC-1α protein in the gastrocnemius of Apc^Min/+ mice. C) Cytochrome C, D) CoxIV and E) PGC-1α protein expression normalized to sedentary mice treated with the control vector. F) Upper - representative Western blot of 4-hydroxynonenal (4HNE)-modified proteins; lower - 4HNE-modified protein expression normalized to sedentary control mice. Values are means ± SE. Significance was set at P <0.05. CC, cage control; ME, main effect;. Wt, wild-type. * Signifies the difference from cage control vector mice.

Figure 6

Exercise training reduces IL-6 induced alterations in mitochondria dynamics, apoptosis and FoxO phosphorylation. A) Representative Western blot of Mfn1, Mfn2 and FIS1 protein in the gastrocnemius of Apc^Min/+ mice. B) Mfn1, C) Mfn2 and D) FIS1 protein expression normalized to sedentary mice treated with the control vector. E) upper - Representative Western blot of total and phosphorylated FoxO proteins; lower - ratio of phosphorylated to total FoxO protein expression normalized to cage control mice. F) Bax mRNA expression. Data are normalized to cage control mice. Values are means ± SE. Significance was set at P <0.05. CC, cage control; ME, main effect; Wt, wild-type. *Signifies the difference from cage control vector mice.

Figure 7

Autophagy and ubiquitin dependent proteolysis are induced with IL-6 over-expression and attenuated with exercise training. A) Representative Western blot of Atg5, Beclin1 and LC3β protein in the gastrocnemius of Apc^Min/+ mice. B) Atg5, C) Beclin1 and D) LC3β protein expression normalized to sedentary cage control mice. E) Atrogin1 mRNA. F) C2 and G) C7 proteasomal subunit mRNA. Data are normalized to cage control groups for wild-type and Apc^Min/+ mice. Values are means ± SE. Significance was set at P <0.05. *Signifies the difference from cage control vector mice. † Signifies the difference within IL-6 treatment.

Figure 8

IL-6 treatment on C2C12 myotubes induces FIS1 expression and a reduction in PGC-1α mRNA. A) Representative Western blot of FIS1, Cytochrome C and CoxIV protein in C2C12 myotubes treated with 0, 20 and 100 ng/ml of IL-6. B) FIS1, C) Cytochrome C and D) CoxIV C protein expression and PGC-1α mRNA E) expression normalized to C2C12 myoblasts treated with vehicle control (0 ng/ml IL-6). Values are means ± SE. Significance was set at P <0.05. *Signifies the difference from 0 ng/ml control.