Altered Activation of Inflammatory Signaling with Diet‐induced Insulin Resistance in the Dystrophic Diaphragm
Authors: Krishna Swathy , Echevarria Kenneth G. , Reed Carter H. , Eo Hyeyoon , Valentine Rudy J. , Selsby Joshua T. 
Published: May 2022

Duchenne muscular dystrophy (DMD) affects 1 in 3500 to 5000 boys born worldwide. It is caused by the absence of functional dystrophin, a sarcolemmal protein. Dystrophin deficiency causes progressive muscle degeneration, leading to numerous cellular dysfunctions, ultimately resulting in death due to respiratory or cardiac failure. Although insulin resistance (IR) is a frequently reported comorbidity in boys with DMD, how IR impacts DMD is less studied. Additionally, the compounded effects of IR and dystropathology on cellular pathways, including inflammatory signaling, have not been previously considered. We hypothesized that a high‐fat, high sucrose diet (HF/HSD) would induce IR and cause activation of inflammatory signaling in dystrophic muscles. To address this hypothesis, 7‐week‐old mdx (diseased) and C57 (healthy) mice were fed a control diet (CD) or HF/HSD for 15 weeks, after which tissues were collected and analyzed. Data were compared using two‐way ANOVAs with Tukey adjustments and repeated measures ANOVA, with significance established at p<0.05. Insulin resistance was confirmed in the HF/HSD groups via increased area under the curve following a glucose tolerance test (p<0.05) and decreased rate of glucose disappearance following an insulin tolerance test (ITT; p<0.05). Further, these outcomes also suggest IR in mdx‐CD compared to C57‐CD as glucose clearance was impaired following an ITT. Visceral adipose (peri‐renal and epididymal) was impacted by disease and diet such that it was lower in mdx mice than C57 and increased in HF/HSD compared to CD, with a significant interaction (p<0.05). Of note, the perirenal adipose weights were increased almost 2‐fold in mdx‐HF/HSD compared to mdx‐CD. To probe for changes associated with both IR and DMD in skeletal muscle, we measured markers of inflammatory signaling using western blot. In whole muscle protein from the diaphragm, TLR4, a pattern recognition receptor, was increased approximately 70% by disease (p<0.05) and 40% by HF/HSD (p<0.05). TNFα, a pro‐inflammatory cytokine, had a significant interaction effect, was increased approximately 50% by disease (p<0.05), decreased 28% by diet (p<0.05), and mdx‐CD was higher (p<0.05) than the other groups. NFκB, a transcription factor that regulates inflammatory responses, increased (p<0.05) as a main effect of disease in whole muscle homogenate and nuclear fraction, but was not changed by diet. IκBα, an inhibitor of NFκB activation, and IKKα, an inhibitor of IκBα and promoter of NFκB, were increased by 1.6‐fold and 2‐fold, respectively (p<0.05), as a function of disease but were resistant to changes caused by diet. AP1 was increased approximately 3‐fold (p<0.05) with dystrophin deficiency in whole muscle homogenate but decreased in the nuclear fraction (45%, p<0.05). In summary, we discovered a HF/HSD induced IR in mdx mice, and our data indicate IR may be a fundamental consequence of dystrophin deficiency. Further, counter to our hypothesis, we discovered that a HF/HSD did not promote inflammatory signaling in dystrophin‐deficient skeletal muscle.