Gut ghrelin regulates hepatic glucose production and insulin signaling via a gut-brain-liver pathway

Abstract

Background: Ghrelin modulates many physiological processes. However, the effects of intestinal ghrelin on hepatic glucose production (HGP) are still unclear. The current study was to explore the roles of intestinal ghrelin on glucose homeostasis and insulin signaling in the liver.

Methods: The system of intraduodenal infusion and intracerebral microinfusion into the nucleus of the solitary tract (NTS) in the normal chow-diet rats and pancreatic-euglycemic clamp procedure (PEC) combined with [3-³H] glucose as a tracer were used to analyze the effect of intestinal ghrelin. Intraduodenal co-infusion of ghrelin, tetracaine and Activated Protein Kinase (AMPK) activator (AICAR), or pharmacologic and molecular inhibitor of N-methyl-D-aspartate receptors within the dorsal vagal complex, or hepatic vagotomy in rats were used to explore the possible mechanism of the effect of intestinal ghrelin on HGP.

Results: Our results demonstrated that gut infusion of ghrelin inhibited duodenal AMP-dependent protein kinase (AMPK) signal pathways, increased HGP and expression of gluconeogenic enzymes, and decreased insulin signaling in the liver of the rat. Intraduodenal co-infusion of ghrelin receptor antagonist [D-Lys³]-GHRP-6 and AMPK agonist with ghrelin diminished gut ghrelin-induced increase in HGP and decrease in glucose infusion rate (GIR) and hepatic insulin signaling. The effects of gut ghrelin were also negated by co-infusion with tetracaine, or MK801, an N-methyl-D-aspartate (NMDA) receptor inhibitor, and adenovirus expressing the shRNA of NR1 subunit of NMDA receptors (Ad-shNR1) within the dorsal vagal complex, and hepatic vagotomy in rats. When ghrelin and lipids were co-infused into the duodenum, the roles of gut lipids in increasing the rate of glucose infusion (GIR) and lowering HGP were reversed.

Conclusions: The current study provided evidence that intestinal ghrelin has an effect on HGP and identified a neural glucoregulatory function of gut ghrelin signaling.

Keywords: Duodenum; Ghrelin; Glucose homeostasis; Insulin resistance.

Fig. 1

Gut ghrelin increases liver glucose production through ghrelin-receptor (GHS-R1a). a Photomicrographs of GHS-R1a immunostaining incubated with saline or GHS-R1a antibody in rat duodenal tissues. Arrowheads indicate positive cells for GHS-R1a staining. b Immunofluorescent images of duodenal tissue sections stained for GHS-R1a (green) and ghrelin (red). c Schematic representation of working hypothesis. Ghrelin with or without GHS-R1a antagonist ([D-Lys³]-GHRP-6) was infused through a duodenal catheter. d Experimental procedure and clamp protocol. Duodenal catheter or venous and arterial catheters were implanted on day 1. The pancreatic clamp studies were performed on day 5. e Gut ghrelin decreased GIR in dose dependent manner. f Cumulative GIR during the steady-state of clamp. g HGP. h Suppression of HGP during the clamp period expressed as the percentage reduction from basal HGP. i Glucose uptake. GIR, the rate of glucose infusion; HGP, hepatic glucose production. Data are means ± SEM, ^** P < 0.01 vs. all other groups. (n = 7 for saline or ghrelin treated group; n = 5 per group for all other groups)

Fig. 2

Gut ghrelin increases hepatic glucose production by activating a gut-brain-liver neurocircuitry. a Schematic representation of the working hypothesis. Gut ghrelin was coinfused with tetracaine, which abolishes the ascending neuronal signal to the brain. A subgroup of rats was given MK-801, an NMDA receptor inhibitor, directly into the NTS. In another study, gut ghrelin was infused into rats with HVAG. b Experimental procedure and clamp protocol. Stereotaxic surgeries were conducted on day 1. A duodenal catheter or venous and arterial catheters were implanted on day 7. HVAG was performed immediately before the implantation of the duodenal and vascular catheters. c and d Gut ghrelin infusion decreased GIR and increased HGP. Rats that received tetracaine in the gut, MK-801 in the NTS or HVAG failed to respond to duodenal ghrelin to decrease the GIR and increase HGP. e Suppression of HGP during the clamp expressed as the percentage decrease from basal HGP. f Glucose uptake was unchanged in all groups. NTS, the nucleus of the solitary tract; HVAG, hepatic vagotomy; NMDA, N-methyl- D-aspartate; GIR, glucose infusion rate; HGP, hepatic glucose production. Values are shown as mean ± SEM. *P < 0.05, **P < 0.01, vs. all other groups. (n = 7 for saline or ghrelin treated group; n = 5 per group for all other groups)

Fig. 3

Gut ghrelin infusion disrupts the gut nutrient sensing-related mechanisms. a Schematic representation of the working hypothesis. Lipid with or without ghrelin, or saline was infused through a duodenal catheter. b Experimental procedure and clamp protocol. c-e Gut lipids infusion increased the GIR (c), and decreased HGP (d and e). When duodenal lipid was co-infused with ghrelin, the effects of lipids on GIR and HGP were abolished. f Glucose uptake was unchanged in all groups. g - i The effect of gut ghrelin on glucose homeostasis during fasting-refeeding. g Schematic representation of the experimental design. A duodenal catheter, the internal jugular vein and carotid artery catheters were implanted on day 1. Rats were subjected to a 40 h fasting from 5 p.m. on day 5 until 9 a.m. on day 7. Ten minutes before the completion of the forty-hour fast, rats were infused with intraduodenal saline or ghrelin (n = 5 per group). Rats were refed on regular chow at time 0 min where food intake and blood glucose were monitored for 20 min. h Plasma glucose levels during refeeding. i Cumulative food intake during refeeding. GIR, glucose infusion rate; HGP, hepatic glucose production. Values are shown as mean ± SEM. *P < 0.05, **P < 0.01 vs. saline or other groups

Fig. 4

Gut ghrelin increases HGP through AMPK inhibition. a Schematic of the working hypothesis. b Experimental procedure and clamp protocol. Ghrelin was infused with or without AICAR, an AMPK activator, through a duodenal catheter. c Representative western blots of AMPK in the mucosal layer of the duodenum. d Gut ghrelin infusion decreased the GIR. When duodenal ghrelin was co-infused with AICAR, the effects of ghrelin on GIR were abolished. GIR, glucose infusion rate. Values are shown as mean ± SEM. *P < 0.05, **P < 0.01 vs. other groups. (n = 7 per group)

Fig. 5

Duodenal ghrelin increases hepatic PEPCK, G6Pase and PGC-1α expression through a gut-brain-liver neurocircuitry. Intraduodenal ghrelin infusion in rats increased hepatic PEPCK, G6Pase and PGC-1α mRNA expression (a) and PEPCK protein expression (b). Rats that received tetracaine (a and b), MK-801 in the NTS (c and d) or HVAG (e and f) failed to respond to duodenal ghrelin to increase hepatic PEPCK, G6Pase and PGC-1α expression. NTS, nucleus of the solitary tract; HVAG, hepatic vagotomy. Values are shown as mean ± SEM. ** P < 0.01 vs. other groups

Fig. 6

Gut ghrelin attenuated hepatic insulin signaling through a gut-brain- liver neurocircuitry. Intraduodenal ghrelin infusion decreased the phosphorylation of InsR (a) and AKT (b) in the liver of rats. Rats that received tetracaine (a and b), MK-801 in the NTS (c and d) or HVAG (e and f) failed to respond to duodenal ghrelin to decrease the phosphorylation of InsR (Tyr 1150/1151) and AKT (Ser473) in the liver of rats. InsR, insulin receptor; AKT, protein kinase B; NTS, nucleus of the solitary tract; HVAG, hepatic vagotomy. Values are shown as mean ± SEM. ** P < 0.01 vs. other groups

Fig. 7

A schematic model showing the effects of duodenal ghrelin on hepatic glucose fluxes and insulin signals. Ghrelin binds to its receptor, GHS-R1a, on the duodenum and inhibits mucosal AMPK. This signal is delivered to the NTS by vagus afferent nerve and activates the neurons in hindbrain region and NMDA receptors. Finally, the signal is relayed from the NTS to the liver via the efferent branch of the vagal nerve to increase HGP and inhibit insulin signal