Changes in Subcellular Distribution of n-Octanoyl or n-Decanoyl Ghrelin in Ghrelin-Producing Cells

Abstract

Background: The enzyme ghrelin O-acyltransferase (GOAT) catalyzes the acylation of ghrelin. The molecular form of GOAT, together with its reaction in vitro, has been reported previously. However, the subcellular processes governing the acylation of ghrelin remain to be elucidated.

Methods: Double immunoelectron microscopy was used to examine changes in the relative proportions of secretory granules containing n-octanoyl ghrelin (C8-ghrelin) or n-decanoyl ghrelin (C10-ghrelin) in ghrelin-producing cells of mouse stomachs. The dynamics of C8-type (possessing C8-ghrelin exclusively), C10-type (possessing C10-ghrelin only), and mixed-type secretory granules (possessing both C8- and C10-ghrelin) were investigated after fasting for 48 h or after 2 weeks feeding with chow containing glyceryl-tri-octanoate (C8-MCT) or glyceryl-tri-decanoate (C10-MCT). The dynamics of C8- or C10-ghrelin-immunoreactivity (ir-C8- or ir-C10-ghrelin) within the mixed-type granules were also investigated.

Results: Immunoelectron microscopic analysis revealed the co-existence of C8- and C10-ghrelin within the same secretory granules (mixed-type) in ghrelin-producing cells. Compared to control mice fed standard chow, the ratio of C10-type secretory granules increased significantly after ingestion of C10-MCT, whereas that of C8-type granules declined significantly under the same treatment. After ingestion of C8-MCT, the proportion of C8-type secretory granules increased significantly. Within the mixed-type granules the ratio of ir-C10-ghrelin increased significantly and that of ir-C8-ghrelin decreased significantly upon fasting.

Conclusion: These findings confirmed that C10-ghrelin, another acyl-form of active ghrelin, is stored within the same secretory granules as C8-ghrelin, and suggested that the types of medium-chain acyl-molecules surrounding and available to the ghrelin-GOAT system may affect the physiological processes of ghrelin acylation.

Keywords: decanoyl ghrelin; fasting; ghrelin O-acyltransferase; immunoelectron microscopy; medium-chain triglycerides; octanoyl ghrelin; radioimmunoassay; secretory granules.

Figure 1

Immunohistochemistry for C8-ghrelin (A) or C10-ghrelin (B) in fundi of stomachs in control mice. The density of DAB staining in cells reflecting C10-ghrelin-immunoreactivity (ir-C10-ghrelin) was lower than that of ir-C8-ghrelin (A,B). Immunofluorescence in fundi of stomachs in control mice (C–E). Ir-C8-ghrelin (C) labeled with Alexa 555 (red), and ir-C10-ghrelin (D) labeled with Alexa 488 (green) are observed in the same section. The yellow color in the merged image (E) indicated a co-localization of ir-C8- and ir-C10-ghrelin. Arrowheads indicate ghrelin-positive cells (A,B). Scale bars represent 100 μm (A,B) and represent 25 μm (C–E).

Figure 2

Double immunogold labeling in ghrelin-producing cells in the stomachs of control mice. Large particles of immunogold (20 nm in diameter) or small particles of immunogold (10 nm in diameter) demonstrated immunoreactivity for C8-ghrelin (ir-C8-ghrelin) or C10-ghrelin (ir-C10-ghrelin), respectively. Over 70% of secretory granules in ghrelin-producing cells exhibited both ir-C8-ghrelin and ir-C10-ghrelin (arrowheads). Occasionally (10–20% of the secretory granules), there appeared granules stained only for ir-C8-ghrelin reflected by large particles (double arrows) or ir-C10-ghrelin reflected by small particles of immunogold (single arrow) in ghrelin-producing cells. Scale bar represents 200 nm.

Figure 3

The proportion of secretory granules that possessed immunoreactivity exclusively for C8-ghrelin (C8-type, cross-hatched column), or exclusively for C10-ghrelin (C10-type, dotted column), or for both C8- and C10-ghrelin (mixed-type, filled column) in ghrelin-producing cells of the stomach in control mice fed ad libitum a standard chow (Control), in mice fasted for 48 h (fasted), or in mice after 2-weeks ingestion of glyceryl-tri-octanoate (C8-MCT-fed) or glyceryl-tri-decanoate (C10-MCT-fed). Data were extracted and analyzed from approximately 250 secretory granules of ghrelin-producing cells in each group (n = 5 mice in each group). ^†p < 0.01; ^††p < 0.001 vs. control and fasted. p < 0.001 vs. control, C8-MCT-fed, and fasted. ^#p < 0.001 vs. control and fasted.