Ghrelin, via corticotropin-releasing factor receptors, reduces glucose uptake and increases lipid content in mouse myoblasts cells

Abstract

Ghrelin and the corticotropin-releasing factor (CRF) family are known regulators of cellular metabolism and energy balance. We previously demonstrated that myoblast glucose metabolism is regulated by ghrelin and that this effect is mediated by CRF receptor type 2 (CRF-R2). Here we explored the effect of des-acyl ghrelin, the major circulating isoform of ghrelin, on cellular metabolism in mouse myoblast C2C12 cells, and examined whether CRF family receptors mediate its metabolic effects in muscle cells. C2C12 cells were exposed to des-acyl ghrelin with or without the CRF-R1- and CRF-R2-specific antagonists antalarmin or antisauvagine-30, respectively. Des-acyl ghrelin reduced glucose uptake and expression of the glucose transporter GLUT4, but induced retinol-binding protein 4 (RBP4) expression. Antalarmin and antisauvagine-30 inhibited the induction of glucose uptake by des-acyl ghrelin and its effect on GLUT4 and RBP4 expression. Moreover, treating C2C12 cells with des-acyl ghrelin resulted in cAMP activation in response to the CRF-R1-specific ligand stressin, and the CRF-R2-specific ligand Ucn3. Furthermore, des-acyl ghrelin reduced the expression of uncoupling proteins UCP2 and UCP3. Adding antalarmin or antisauvagine-30 to the medium reversed this effect. Finally, des-acyl ghrelin elevated lipid content and acetyl-CoA carboxylase expression in C2C12 cells. Our results suggest that during food deprivation, des-acyl ghrelin signals the muscle cells that glucose levels are low and that they should switch to fatty acids for their metabolic fuel.

Keywords: C2C12 cell; CRF receptor; des-acyl ghrelin; glucose metabolism; lipid metabolism.

FIGURE 1

CRF‐R1 and CRF‐R2 inhibitors block des‐acyl ghrelin inhibition of glucose uptake. Des‐acyl ghrelin decreases H³‐deoxy‐glucose uptake into C2C12 cells (a). Antalarmin, a CRF‐R1‐specific antagonist (b), and antisauvagine‐30, a CRF‐R2‐specific antagonist (c), inhibit des‐acyl ghrelin‐induced glucose uptake. The examples shown are representative of at least three experiments. *p < .05 versus control

FIGURE 2

Des‐acyl ghrelin increases cAMP levels in C2C12 cells treated with CRF‐R1‐ or CRF‐R2‐selective ligands. (a) Stressin‐stimulated and (b) Ucn3‐stimulated cAMP accumulation in C2C12 cells pretreated with des‐acyl ghrelin. The examples shown are representative of at least three experiments. *p < .05 versus control

FIGURE 3

Des‐acyl ghrelin reduces GLUT4 levels in C2C12 cells. Des‐acyl ghrelin reduces GLUT4 mRNA (a) and protein (b) expression levels in C2C12 cells. The examples shown are representative of at least three experiments. *p < .05 versus control

FIGURE 4

Des‐acyl ghrelin decreases RBP4 levels in C2C12 cells. Prolonged exposure to des‐acyl ghrelin upregulates RBP4 mRNA expression levels (a) in a dose‐dependent manner (b). Antalarmin, a CRF‐R1‐specific antagonist (c), and antisauvagine‐30, a CRF‐R2‐specific inhibitor (d), blocked this effect of ghrelin on RBP4 mRNA levels. The examples shown are representative of at least three experiments. *p < .05 versus control

FIGURE 5

Ghrelin upregulates UCP2 and UCP3 mRNA expression in C2C12 cells. Prolonged exposure to ghrelin increases both UCP2 and UCP3 mRNA expression levels in a time‐dependent (a and c, respectively) and dose‐dependent (b and d, respectively) manner. The CRF‐R1‐specific antagonist antalarmin (e, g), and CRF‐R2‐specific inhibitor antisauvagine‐30 (f, h) blocked this effect of ghrelin on both UCP2 and UCP3 mRNA levels. The examples shown are representative of at least three experiments. *p < .05 versus control

FIGURE 6

Des‐acyl ghrelin upregulates lipid content and fatty acid‐related protein expression. (a) Different concentrations of des‐acyl ghrelin were added to the cells for 72 hr, and lipid content assay was performed. (b) Cells were incubated with the indicated concentrations of des‐acyl ghrelin for 72 hr. Then proteins were extracted from the cells and ACC expression levels were examined. (c, d) C2C12 cells were incubated with des‐acyl ghrelin, and antalarmin (c) or antisauvagine‐30 (d) was added to the medium; lipid content was measured according to the protocol described in Materials and Methods. The examples shown are representative of at least three experiments. *p < .05 versus control