The effects of diet and chronic exercise on skeletal muscle ghrelin response

Abstract

Background: Recent findings indicate that ghrelin, particularly the unacylated form (UnAG), acutely stimulates skeletal muscle fatty acid oxidation (FAO) and can preserve insulin signaling and insulin-stimulated glucose uptake in the presence of high concentrations of saturated fatty acids. However, we recently reported that the stimulatory effect of ghrelin on FAO and subsequent ability to protect insulin stimulated glucose uptake was lost following 6-weeks (6w) of chronic high fat feeding. In the current study we examined the effects of both short-term 5 day (5d) and chronic 6w high-fat diet (HFD) on muscle ghrelin response, and whether exercise training could prevent the development of muscle ghrelin resistance with 6w of HFD.

Methods and results: Soleus muscle strips were isolated from male rats to determine the direct effects of acylated (AG) and UnAG isoforms on FAO and glucose uptake. A 5d HFD did not alter the response of soleus muscle to AG or UnAG. Conversely, 6w of HFD was associated with a loss of ghrelin's ability to stimulate FAO and protect insulin stimulated glucose uptake. Muscle response to UnAG remained intact following the 6w HFD with chronic exercise training. Unexpectedly, muscle response to both AG and UnAG was also lost after 6w of low-fat diet (LFD) consumption. Protein content of the classic ghrelin receptor, GHS-R1a, was not affected by diet or training. Corticotropin-releasing hormone receptor-2 (CRF-2R) content, a putative receptor for ghrelin in muscle, was significantly decreased in soleus from 6w HFD-fed animals and increased following exercise training. This may explain the protection of UnAG response with training in HFD-fed rats but does not explain why ghrelin response was also lost in LFD-fed animals.

Conclusions: UnAG protects muscle glucose uptake during acute lipid oversupply, likely due to its ability to stimulate FAO. This effect is lost in 6w HFD-fed animals but protected with exercise training. Unexpectedly, ghrelin response was lost in 6w LFD-fed animals. The loss of ghrelin response in muscle with a LFD cannot be explained by a change in putative ghrelin receptor content. We believe that the sedentary nature of the animals is a major factor in the development of muscle ghrelin resistance and warrants further research.

Keywords: Exercise training; Ghrelin; Glucose transport; High-fat diet; Lipid oxidation; Skeletal muscle.

Fig. 1

Muscle fatty acid oxidation following 5d diets. Rates of palmitate oxidation in isolated soleus muscle from 5d LFD and HFD animals following medium palmitate (MP; 1 mM) or high palmitate (HP; 2 mM) exposure +/- AG/UnAG (150 ng/ml) treatment. Data were analyzed using a repeated measures two-way ANOVA (n = 8 for each) and expressed as individual data points and the mean ± standard deviation. Data sharing a letter are not statistically significant from each other within each diet condition.

Fig. 2

Body weight, caloric intake, and blood glucose measurements following 6w of dietary and exercise interventions. Food intake (Fig. 2A), caloric intake (Fig. 2B), body weights (Fig. 2C), blood glucose concentrations (Fig. 2D) and glucose AUC (Fig. 2E) following LFD-SED, HFD-SED, and HFD-EX interventions. Body weight, food intake, caloric intake (n = 17–19/group) and blood glucose tolerance data (n = 9–11/group) were analyzed using a repeated measures two-way ANOVA. Data are expressed as mean ± standard deviation. Asterisks denote significant differences between groups: * indicates a difference between HFD-SED and LFD-SED animals (Fig. 2D), ** indicate differences between HFD-SED in comparison to both HFD-EX and LFD-SED animals (Fig. 2B; Fig. 2C) and *** indicates significant differences between all groups. For AUC (Fig. 2E), data not sharing a letter are statistically significant from each other.

Fig. 3

Muscle fatty acid oxidation following 6w of dietary and exercise interventions. Rates of palmitate oxidation in isolated soleus muscle from LFD-SED, HFD-SED and HFD-EX animals following low-palmitate (LP; 0.2 mM) or high-palmitate (HP; 2.0 mM) exposure +/- AG/UnAG (150 ng/ml) ghrelin treatment. Data were analyzed using a repeated measures two-way ANOVA (n = 9 for each group) and expressed as individual data points and the mean ± standard deviation. p < 0.05 was considered statistically significant. Data sharing a letter are not statistically significant from each other within each diet condition.

Fig. 4

Muscle glucose uptake following 6w of dietary and exercise interventions. Rates of insulin (INS; 10mU/ml) stimulated glucose uptake in isolated soleus muscle from LFD-SED, HFD-SED and HFD-EX animals following low palmitate (LP; 0.2 mM) or high palmitate (HP; 2.0 mM) exposure +/- AG/UnAG (150 ng/ml) ghrelin treatment. Data were analyzed using a repeated measures two-way ANOVA (n = 8 for LFD-SED and HFD-EX; n = 13 for HFD-SED) and expressed as individual data points and the mean ± standard deviation. p < 0.05 was considered statistically significant. Data sharing a letter are not statistically significant from each other within each diet condition.

Fig. 5

Training adaptations following 6w of dietary and exercise interventions. Cytochrome c oxidase subunit 4 (COX IV), Citrate synthase (CS), and peroxisome proliferator-activated receptor coactivator 1-alpha (PGC-1α) protein expression in isolated soleus of LFD-SED, HFD-SED, and HFD-EX animals. Data were analyzed using an ordinary one-way ANOVA (n = 8–16 for each) and expressed as individual data points and the mean ± standard deviation. p < 0.05 was considered statistically significant. Data sharing a letter are not statistically significant from each other.

Fig. 6

Effects of 6w dietary and exercise interventions on muscle substrate transporters. GLUT 4 and FAT/CD36 protein expression in isolated soleus of LFD-SED, HFD-SED, and HFD-EX animals. Data were analyzed using an ordinary one-way ANOVA (n = 8/group) and expressed as individual data points and the mean ± standard deviation. p < 0.05 was considered statistically significant. Data sharing a letter are not statistically significant from each other.

Fig. 7

Effects of 6w dietary and exercise interventions on ghrelin receptors. Growth hormone secretagogue receptor type 1 (GHS-R1) and Corticotropin releasing factor receptor 2 (CRF-2R) protein expression in isolated soleus of LFD-SED, HFD-SED, and HFD-EX animals. Data were analyzed using an ordinary one-way ANOVA (n = 8–14 per group) and expressed as individual data points and the mean ± standard deviation. p < 0.05 was considered statistically significant. Data sharing a letter are not statistically significant from each other.

Fig. 8

Graphical representation of acylated (AG) and unacylated (UnAG) ghrelin signalling in isolated rat skeletal muscle during acute lipid oversupply, and the effects on insulin stimulated glucose uptake and palmitate oxidation. This figure serves as a representative schematic; thus, not everything included in the figure was assessed in the current study. Schematic represents sedentary animals and chronic treadmill trained animals on a high-fat diet (HFD). Sedentary animals on a low-fat diet shared a similar metabolic profile to sedentary animals on a HFD and were not included in this figure. Dashed lines and question mark represent unassessed mechanisms. Upward arrow indicates increase. Downward arrow indicates inhibition. Broken arrow indicates loss of signaling pathway. GHS-R1; Growth Hormone Secretagogue Receptor 1, CRF-2R; Corticotropin-Releasing Factor 2 Receptor, FAT/CD36; Fatty Acid Translocase, FABPpm; Fatty Acid Binding Protein plasma membrane, IR; Insulin Receptor, GLUT4; Glucose Transporter 4, DAG; diacylglycerol. This figure was created with use of free images from smart.servier.com.