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. 2013 Sep;27(9):3572-82.
doi: 10.1096/fj.13-230375. Epub 2013 Jun 3.

Cancer- and endotoxin-induced cachexia require intact glucocorticoid signaling in skeletal muscle

Theodore P Braun  1 , Aaron J Grossberg, Stephanie M Krasnow, Peter R Levasseur, Marek Szumowski, Xin Xia Zhu, Julia E Maxson, J Gabriel Knoll, Anthony P Barnes, Daniel L Marks
Affiliations

Cancer- and endotoxin-induced cachexia require intact glucocorticoid signaling in skeletal muscle

Theodore P Braun et al. FASEB J. 2013 Sep.

Abstract

Cachexia is a wasting condition defined by skeletal muscle atrophy in the setting of systemic inflammation. To explore the site at which inflammatory mediators act to produce atrophy in vivo, we utilized mice with a conditional deletion of the inflammatory adaptor protein myeloid differentiation factor 88 (MyD88). Although whole-body MyD88-knockout (wbMyD88KO) mice resist skeletal muscle atrophy in response to LPS, muscle-specific deletion of MyD88 is not protective. Furthermore, selective reexpression of MyD88 in the muscle of wbMyD88KO mice via electroporation fails to restore atrophy gene induction by LPS. To evaluate the role of glucocorticoids as the inflammation-induced mediator of atrophy in vivo, we generated mice with targeted deletion of the glucocorticoid receptor in muscle (mGRKO mice). Muscle-specific deletion of the glucocorticoid receptor affords a 71% protection against LPS-induced atrophy compared to control animals. Furthermore, mGRKO mice exhibit 77% less skeletal muscle atrophy than control animals in response to tumor growth. These data demonstrate that glucocorticoids are a major determinant of inflammation-induced atrophy in vivo and play a critical role in the pathogenesis of endotoxemic and cancer cachexia.

Keywords: MyD88; adenocarcinoma; atrophy; corticosterone; inflammation; lipopolysaccharide; sickness behavior.

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Figures

Figure 1.
Figure 1.
MyD88 is required for LPS-induced muscle atrophy. WT and wbMyD88KO mice (n=5–7/group) were treated with LPS (1 mg/kg) immediately prior to lights off, and euthanized 18 h later. A, B) Food intake (A) and body weight (B) were measured. C) Gastrocnemius muscle was weighed and normalized to pretreatment body weight and presented as weight loss relative to vehicle-injected control mice of the same genotype. D) Muscle gene expression measured by quantitative real-time PCR with GAPDH as an endogenous control. V, vehicle; L, LPS; KO, wbMyD88KO. *P < 0.05, **P < 0.01, ***P < 0.001; 2-way ANOVA with Bonferroni post hoc test or t test as appropriate.
Figure 2.
Figure 2.
Muscle-specific deletion of MyD88 fails to prevent atrophy gene induction by LPS. Expression of MAFbx, MuRF-1, and Foxo1 (A), KLF15, REDD1, and Mstn (B), and Bnip3, CstL, and Gabarapl1 (C) in the gastrocnemius muscle of female mMyD88KO mice (n=6–10/group) 8 h after i.p. LPS injection (250 μg/kg), measured by quantitative real-time PCR using GAPDH as an endogenous control. V, vehicle, L, LPS. ***P < 0.001; 2-way ANOVA with Bonferroni post hoc test.
Figure 3.
Figure 3.
Muscle-specific deletion of MyD88 fails to prevent muscle atrophy in response to LPS. Male mMyD88KO mice (n=7–11/group) were treated with LPS (1 mg/kg) immediately prior to lights off, and euthanized 18 h later. A, B) Food intake (A) and body weight (B) were measured. C) Gastrocnemius muscle was weighed and normalized to pretreatment body weight; results are presented as weight loss relative to vehicle-injected control mice of the same genotype. D) Muscle gene expression, measured by quantitative real-time PCR with GAPDH as an endogenous control. V, vehicle; L, LPS; KO, mMyD88KO. ***P < 0.001; 2-way ANOVA with Bonferroni post hoc test or t test as appropriate.
Figure 4.
Figure 4.
MyD88 signaling in muscle is not sufficient for the induction of MAFbx by LPS. Wild type (n=2/group) and wbMyD88KO (n=4/group) mice were electroporated in the TA muscle with plasmid containing mCherry-IRES-GFP or MyD88-IRES-GFP and then treated with LPS (250 μg/kg). MAFbx mRNA levels were examined by in situ hybridization in regions expressing GFP in serial sections 8 h after LPS treatment. For ease of comparison between GFP and in situ hybridization sections, connective tissue boundaries and section edges have been outlined. Scale bars = 400 μm.
Figure 5.
Figure 5.
Muscle-specific deletion of the GR prevents muscle atrophy in response to pharmacologic dexamethasone treatment. GRLox/Lox and mGRKO mice (n=5 or 6/group) were treated for 3 d with dexamethasone (5 mg/kg). A, B) Food intake (A) and body weight (B) were measured daily. C) Gastrocnemius muscle was weighed and normalized to pretreatment body weight; results are presented as weight loss relative to vehicle-injected control mice of the same genotype. D) Gastrocnemius muscle gene expression, measured by quantitative real-time PCR with GAPDH as an endogenous control. V, vehicle; D, dexamethasone; KO, mGRKO. *P < 0.05, **P < 0.01, ***P < 0.001; 2-way ANOVA with Bonferroni post hoc test or t test as appropriate.
Figure 6.
Figure 6.
Muscle-specific deletion of the GR prevents atrophy gene induction by LPS. Expression of MAFbx, MuRF-1, and Foxo1 (A), KLF15, REDD1, and Mstn (B), and Bnip3, CstL, and Gabarapl1 (C) in the gastrocnemius muscle of female mGRKO mice (n=6–9/group) 8 h after i.p. LPS injection (250 μg/kg), as measured by quantitative real-time PCR using GAPDH as an endogenous control. V, vehicle, L, LPS. *P < 0.05, **P < 0.01, ***P < 0.001; 2-way ANOVA with Bonferroni post hoc test.
Figure 7.
Figure 7.
Muscle-specific deletion of the GR attenuates muscle atrophy in response to LPS. Male mGRKO (n=6–9/group) were treated with LPS (1 mg/kg) immediately prior to lights off, and euthanized 18 h later. A subset of vehicle-treated animals was pair fed to the LPS treatment group. A, B) Food intake (A) and body weight (B) were measured. C) Gastrocnemius muscle was weighed and normalized to pretreatment body weight and presented as weight loss relative to vehicle-injected control mice of the same genotype. D) Muscle gene expression measured by quantitative real-time PCR with GAPDH as an endogenous control. E) TA muscles were cut in cross section and immunostained with antibodies against laminin, myosin IIb, myosin IIa, and myosin I. Type IIb, IId/x (absent IIb, IIa, or I staining), and IIa fiber area was measured. F) Representative images of TA in a region of type IIb fibers (white, laminin; green, myosin IIb). Scale bars = 50 μm. V, vehicle, L, LPS, P, pair fed. *P < 0.05, **P < 0.01, ***P < 0.001; 2-way ANOVA with Bonferroni post hoc test.
Figure 8.
Figure 8.
Muscle-specific deletion of the GR attenuates muscle atrophy in the setting of cancer cachexia. Female GRLox/Lox and mGRKO mice (n=10–15/group) were injected with LLC cells or were sham-treated. A) Tumor mass was measured. B) Gastrocnemius muscle was weighed and normalized to pretreatment body weight; results are presented as weight loss relative to sham-treated control mice of the same genotype. C) Muscle gene expression measured by quantitative real time PCR with GAPDH as an endogenous control. D) Representative images of TA muscles that were cut in cross section and immunostained with antibodies against laminin (white) and myosin IIb (green). E) Fiber CSA of myosin IIb, IIa, and IId/x fibers normalized to initial body weight. Scale bars = 50 μm. S, sham, T, tumor. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. sham-treated mice of the same genotype; t test or 2-way ANOVA with Bonferroni post hoc test as appropriate.
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