Mechanistic Investigation of GHS-R Mediated Glucose-Stimulated Insulin Secretion in Pancreatic Islets

Abstract

Ghrelin receptor, a growth hormone secretagogue receptor (GHS-R), is expressed in the pancreas. Emerging evidence indicates that GHS-R is involved in the regulation of glucose-stimulated insulin secretion (GSIS), but the mechanism by which GHS-R regulates GSIS in the pancreas is unclear. In this study, we investigated the role of GHS-R on GSIS in detail using global Ghsr^-/- mice (in vivo) and Ghsr-ablated pancreatic islets (ex vivo). GSIS was attenuated in both Ghsr^-/- mice and Ghsr-ablated islets, while the islet morphology was similar between WT and Ghsr^-/- mice. To elucidate the mechanism underpinning Ghsr-mediated GSIS, we investigated the key steps of the GSIS signaling cascade. The gene expression of glucose transporter 2 (Glut2) and the glucose-metabolic intermediate-glucose-6-phosphate (G6P) were reduced in Ghsr-ablated islets, supporting decreased glucose uptake. There was no difference in mitochondrial DNA content in the islets of WT and Ghsr^-/- mice, but the ATP/ADP ratio in Ghsr^-/- islets was significantly lower than that of WT islets. Moreover, the expression of pancreatic and duodenal homeobox 1 (Pdx1), as well as insulin signaling genes of insulin receptor (IR) and insulin receptor substrates 1 and 2 (IRS1/IRS2), was downregulated in Ghsr^-/- islets. Akt is the key mediator of the insulin signaling cascade. Concurrently, Akt phosphorylation was reduced in the pancreas of Ghsr^-/- mice under both insulin-stimulated and homeostatic conditions. These findings demonstrate that GHS-R ablation affects key components of the insulin signaling pathway in the pancreas, suggesting the existence of a cross-talk between GHS-R and the insulin signaling pathway in pancreatic islets, and GHS-R likely regulates GSIS via the Akt-Pdx1-GLUT2 pathway.

Keywords: glucose transporter 2 (Glut2); glucose-stimulated insulin secretion (GSIS); growth hormone secretagogue receptor (GHS-R); insulin; islets; pancreatic and duodenal homeobox 1 (Pdx1).

Figure 1

GHS-R ablation decreases glucose-stimulated insulin secretion (GSIS). (A) 4.5-month-old WT and Ghsr^−/− mice were fasted overnight, and GSIS was performed in vivo, n = 5. (B) Islets from 4-month-old WT and Ghsr^−/− mice were treated with 3.3, 11.1 or 22. 2 mM glucose for 2 h. Insulin content was measured and normalized to the total protein content of islets, n = 5–8. (C) H&E staining and immunostaining of insulin and glucagon of pancreatic sections from WT and Ghsr^−/− mice. (D) Ins1 and Ins2 gene expression was measured from islets of 6.5-month-old mice, n = 5. (E) Insulin protein content of the whole pancreas (4 months, n = 5) and islet (5.5 m, n = 8) was measured and normalized by total protein content. * p < 0.05, ** p < 0.001, WT vs. Ghsr^−/−.

Figure 2

Ghsr^−/− islets have a lower ATP/ADP ratio. (A) Islets from WT and Ghsr^−/− mice were treated with 3.3 mM glucose in the presence or absence of 30 mM KCl, and insulin was measured after 2 h. Insulin was normalized to total protein content in islets, n = 4–5. (B) Ratio of mitochondrial DNA to nuclear DNA content in islets. n = 4. (C) 15 Islets/well from WT and Ghsr^−/− mice were treated with 3.3 or 22.2 mM glucose for 2 h, and then the ATP/ADP ratio was measured, n = 6–8. (D) OCR was assessed in islets of WT and Ghsr^−/− mice. Islets of WT and Ghsr^−/− mice were pretreated with 3.3 or 22.2 mM glucose for 2 h, and then the basal OCR rate was measured. (E) Change in OCR from baseline after 22.2 mM glucose treatment. (F) ATP turnover, n = 3–5. * p < 0.05, WT vs. Ghsr ^−/−; ** p < 0.001, WT vs. Ghsr^−/−; # p < 0.05, 3.3 mM glucose vs. 22.2 mM glucose (WT).

Figure 3

Glucose uptake is reduced in Ghsr^−/− mice. (A) Glut2 gene expression was measured in islets of 6.5-month-old WT and Ghsr^−/− mice, n = 5. (B) G6P was measured from the pancreas of 4-month-old WT and Ghsr^−/− mice, which were fasted overnight, injected with glucose and then sacrificed 60 min later, n = 7–9. (C) Schematic diagram of impairments in GSIS pathway in Ghsr-ablated β-cells. Impaired Glut2 expression leads to the reduced formation of G6P, contributing to the lower generation of ATP, resulting in attenuated insulin secretion. * p < 0.05, ** p < 0.001, WT vs. Ghsr^−/−.

Figure 4

Genes of IR-IRS1/IRS2 pathway and transcription factor Pdx1 are downregulated in Ghsr^−/− mice. (A) Gene expression of IR, IRS1, and IRS2 were measured in islets of 6.5-month-old mice, n = 5. (B) Protein expression of p-AKT and t-AKT was measured in the pancreas from 12–14-month-old WT and Ghsr^−/− mice injected with saline or insulin. (C) Gene expression of transcription factors, Pdx1, and MafA were measured in islets of 6.5-month-old mice, n = 5. (D) Protein expression of Pdx1 was measured in the pancreas of 5-month-old WT and Ghsr^−/− mice, n = 4. (E) Schematic diagram illustrating that GHS-R regulates GSIS via the AKt-Pdx1 pathway. The numbers in the diagrams are denoted as below: (1) GHS-R ablation in β-cells downregulates Pdx1 and MafA either directly or indirectly reducing the expression of insulin signaling of IR, IRS1, IRS2, and Akt; (2) The downregulation of Pdx1 and MafA impairs Glut2 expression; (3) Reduced Glut2 expression leads to decreased glucose uptake; (4) Reduced generation of Glucose-6-phosphate leads to lower ATP/ADP ratio; (5) Reduced ATP/ADP ratio causes partial closing of ATP sensitive K_ATP channels (6), which leads to insufficient membrane depolarization (7); This ultimately results in a reduced influx of Ca²⁺ ions (8) that suppresses insulin secretion (9). ** p < 0.001, WT vs. Ghsr^−/−.