Unacylated ghrelin stimulates fatty acid oxidation to protect skeletal muscle against palmitate-induced impairment of insulin action in lean but not high-fat fed rats

Abstract

Background: Ghrelin is a gut hormone that spikes in circulation before mealtime. Recent findings suggest that both ghrelin isoforms stimulate skeletal muscle fatty acid oxidation, lending to the possibility that it may regulate skeletal muscle's handling of meal-derived substrates. It was hypothesized in the current study that ghrelin may preserve muscle insulin response during conditions of elevated saturated fatty acid (palmitate) availability by promoting its oxidation.

Methods and results: Soleus muscle strips were isolated from male rats to determine the direct effects of ghrelin isoforms on fatty acid oxidation, glucose uptake and insulin signaling. We demonstrate that unacylated ghrelin (UnAG) is the more potent stimulator of skeletal muscle fatty acid oxidation. Both isoforms of ghrelin generally protected muscle from impaired insulin-mediated phosphorylation of AKT Ser⁴⁷³ and Thr³⁰⁸, as well as downstream phosphorylation of AS160 Ser⁵⁸⁸ during high palmitate exposure. However, only UnAG was able to preserve insulin-stimulated glucose uptake during exposure to high palmitate concentrations. The use of etomoxir, an irreversible inhibitor of carnitine palmitoyltransferase (CPT-1) abolished this protection, strongly suggesting that UnAG's stimulation of fatty acid oxidation may be essential to this protection. To our knowledge, we are also the first to investigate the impact of a chronic high-fat diet on ghrelin's actions in muscle. Following 6 wks of a high-fat diet, UnAG was unable to preserve insulin-stimulated signaling or glucose transport during an acute high palmitate exposure. UnAG was also unable to further stimulate 5' AMP-activated protein kinase (AMPK) or fatty acid oxidation during high palmitate exposure. Corticotropin-releasing hormone receptor-2 (CRF-2R) content was significantly decreased in muscle from high-fat fed animals, which may partially account for the loss of UnAG's effects.

Conclusions: UnAG is able to protect muscle from acute lipid exposure, likely due to its ability to stimulation fatty acid oxidation. This effect is lost in high-fat fed animals, implying a resistance to ghrelin at the level of the muscle. The underlying mechanisms accounting for ghrelin resistance in high fat-fed animals remain to be discovered.

Keywords: Ghrelin; Glucose transport; High-fat diet; Insulin action; Lipid oxidation; Rat; Skeletal muscle.

Fig. 1

Insulin Signaling: The activation of insulin signaling protein AKT at phosphorylation sites Ser⁴⁷³ and Thr³⁰⁸ as well as AS160 at site Ser⁵⁸⁸ in isolated soleus muscle following muscle exposure to low (LP; 0.2mM) or high (HP; 2mM) palmitate concentrations with or without AG/UnAG (150ng/ml) and insulin (10mU/ml). Data were analyzed using a repeated measure one-way ANOVA (n = 8-11) and expressed as individual data points and the mean ± standard error, in arbitrary protein units (phospho/total). Data sharing a letter are not statistically different from each other. P < 0.05 was considered statistically significant.

Fig. 2

Glucose Uptake and Palmitate Oxidation: Rates of basal and insulin-stimulated (10mU/ml) glucose uptake (A) and palmitate oxidation (B) following the exposure of isolated soleus muscle to either low (LP) or high (HP) palmitate concentrations with or without AG/UnAG (150ng/ml). Data were analyzed using a repeated measure one-way ANOVA (n=8 for each) and expressed as individual data points and the mean ± standard error. Data sharing a letter are not statistically different from each other. P < 0.05 was considered statistically significant.

Fig. 3

Inhibition of Fatty Acid Oxidation: The effects of etomoxir (CPT-1 inhibitor) dose (A) and exposure time (B) or vehicle (DMSO) on rates of palmitate oxidation in isolated soleus muscle during an exposure to 2mM palmitate. Rates of insulin-stimulated (10mU/ml) glucose transport (C) were also assessed following 4 h low (LP) or high (HP) palmitate exposure with or without AG/UnAG (150ng/ml) in the presence or absence of 100µM etomoxir (4 h). Data were analyzed using a repeated measure one-way ANOVA (n = 6-8) and expressed as individual data points and the mean ± standard error. Data sharing a letter are not statistically different from each other. P < 0.05 was considered statistically significant.

Fig. 4

Whole-Body Measurements: Food (A) and energy (B) intake as well as body weights (C) during a 6-week dietary intervention providing rats with diet either low (10%) or high (60%) in kcals from fat, ad libitum. Following an overnight fast, blood glucose response was tracked following an IPGTT (D) at the end of 6-weeks on specified diet. Food, energy intake and body weight data were analyzed using a two-way (main effects and interaction displayed in text) ANOVA (n=8-12). Glucose AUC data were analyzed using an unpaired t test (LFD: n=7; HFD: n=12). All data were expressed as individual data points and the mean ± standard error. An asterisk (*) depicts a significant difference at a given week between dietary groups (main effect; HFD vs. LFD). P < 0.05 was considered statistically significant.

Fig. 5

Glucose Uptake and Palmitate Oxidation in High-Fat Fed Animals: Rates of palmitate oxidation (A) and basal or insulin-stimulated (10mU/ml) glucose transport (B) following either low (LP) or high (HP) palmitate exposure either with or without AG/UnAG, in isolated soleus muscle from 6-week high-fat fed animals. Data were analyzed using a repeated measure one-way ANOVA (n=10-12) and were expressed as individual data points and the mean ± standard error. Data sharing a letter are not statistically different from each other. P < 0.05 was considered statistically significant.

Fig. 6

Muscle Signaling in High-Fat Fed Animals: The phosphorylation (activation) of the cellular energy-sensing protein AMPK (Thr¹⁷²) and its downstream target ACC (Ser⁷⁹) following low (LP) or high (HP) palmitate exposure either with or without AG/UnAG treatment, in isolated soleus muscle from 6-week high fat-fed rats. Data were analyzed using a repeated measure one-way ANOVA (n=11-12) and were expressed as individual data points and the mean ± standard error. Also shown, are CRF-2R and GHS-R1 receptors, as well as the fatty acid transporter FABPpm from soleus muscle of the same 6-week high-fat fed rats. Data were analyzed using an unpaired t test (n=7-12) and were expressed as individual data points and the mean ± standard error. Data sharing a letter are not statistically different from each other. P < 0.05 was considered statistically significant.