The Effect of Glucose Administration on Yes‐associated Protein (YAP) Activity in Healthy and Cachectic Mouse Muscles

Abstract

Yes‐associated protein (YAP) is a downstream effector of Hippo signaling and regulates skeletal muscle size and homeostasis. The change of YAP activity has been reported in aging and dystrophic muscles. Our preliminary data showed that tumor burden alters the YAP activity in skeletal muscle. Recent studies showed that glucose metabolism also regulates YAP activity in cancer cells; however, it is unknown whether glucose administration stimulates YAP activity in skeletal muscle. The purpose of this study was to determine whether the YAP activity would change after acute glucose administration in healthy and cachectic mouse muscles. We hypothesized that glucose could stimulate YAP activity, at least in healthy muscle. Approximately 4‐month‐old wild‐type (WT, n=10) and ApcMin/+ mice (Min, n=10) were used in this study. Each group of mice was assigned to either control (Con, n=5 per group) or glucose administration (Glu n=5 per group) treatment. At first, all the mice fasted for four hours. Glucose dissolved in phosphate‐buffered saline (PBS, 2 mg/kg) was administered intraperitoneally to Glu groups. Con groups received PBS injections. Thirty minutes after glucose administration, the mice were euthanized, and plantaris muscles (PLAN) were removed. The time to sacrifice was based on the peak insulin secretion during a glucose tolerance test. To ensure injection accuracy, the glucose levels were measured using a handheld glucometer before glucose injection and at death. After total protein was extracted, routine Western blotting was performed using 50~100 µg of the total protein. Each phosphorylated protein was normalized to its respective total protein. Unpaired t‐test (WT vs. Min) and a two‐way ANOVA (genotype x glucose treatment) were used for statistical analysis. The levels of significance were set at 0.05. The body weight of Min mice was smaller than that of WT mice (19.8±1.2 mg vs. 17.3±0.5 mg for WT and Min, respectively, p=0.05). Likewise, the PLAN muscle mass of Min mice was smaller than that of WT mice (19.8±1.2 mg vs. 17.3±0.5 mg for WT and Min, respectively, p=0.05). There was no significant difference in fasting glucose levels between WT and Min mice, and glucose administration increased plasma glucose levels regardless of genotype (main effect of glucose administration, p<0.05). The activity levels of glycogen synthase kinase 3 beta (GSK3β) were comparable at the baseline (1.1±0.1 vs. 1.1±0.2 for WT and Min, respectively) and after glucose administration (1.1±0.2 vs. 1.1±0.4 for WT and Min, respectively). Min mice showed a reduction in total ribosomal protein S6 level after fasting (1.0±0.2 vs. 0.4±0.2 for WT and Min, respectively, p<0.05) and in response to glucose administration (2.7±1.5 vs. 0.6±0.4 for WT and Min, respectively, p<0.05). Interestingly, the basal YAP levels were comparable between WT and Min mice (1.0±0.1 vs. 1.1±0.1 for WT and Min, respectively), and the glucose administration did not alter the YAP levels (1.1±0.1 vs. 1.1±0.2 for WT and Min, respectively). These data suggest that YAP activity is independent of acute glucose administration in healthy and cachectic mouse skeletal muscles.