Lipopolysaccharide differentially decreases plasma acyl and desacyl ghrelin levels in rats: potential role of the circulating ghrelin-acylating enzyme GOAT

Abstract

Bacterial lipopolysaccharide (LPS) in rodents is an established model for studying innate immune responses to gram-negative bacteria and mimicking symptoms of infections including reduced food intake associated with decreased circulating total ghrelin levels. The ghrelin-acylating enzyme, ghrelin-O-acyltransferase (GOAT) involved in the formation of acyl ghrelin (AG) was recently identified. We investigated changes in circulating AG, desacyl ghrelin (DG) and GOAT induced by intraperitoneal LPS (100 microg/kg) and associated changes in food intake. Plasma AG and total ghrelin were assessed by radioimmunoassay, GOAT protein by Western blot and mRNA by RT-qPCR. DG was derived from total minus AG. Plasma AG and DG were decreased at 2, 5 and 7 h (p<0.01) post-injection compared to vehicle and recovered at 24 h. At 2 h there was a significantly greater decrease of AG (-53%) than DG (-28%) resulting in a decreased AG/DG ratio (1:5, p<0.01), which thereafter returned to pre-injection values (1:3). This altered ratio was associated with a 38% decrease in plasma GOAT protein compared to vehicle (p<0.001), whereas gastric GOAT protein was slightly increased by 10% (p<0.05). GOAT mRNA expression was unchanged. Food intake was reduced by 58% measured during the 1.5-2 h period post-LPS injection. Decreased plasma AG and DG preceded the rise in rectal temperature and blood glucose that peaked at 7 h. These data indicate that LPS induces a long-lasting reduction of AG and DG levels that may have a bearing with the decrease in food intake. The faster drop in AG than DG within 2 h is associated with reduced circulating GOAT.

Fig. 1

LPS reduces plasma concentrations of acyl and desacyl ghrelin in conscious rats. LPS (100 μg/kg body weight) or vehicle was injected ip during the light phase in overnight fasted rats chronically implanted with a jugular catheter. Blood was withdrawn before and 2h, 5h, 7h and 24h post injection. Acyl ghrelin (A) and total ghrelin levels were assessed by radioimmunoassay. Desacyl ghrelin (B) levels were obtained by calculating the difference of total ghrelin minus acyl ghrelin. Each bar represents the mean ± sem of 5 rats/group. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. vehicle at the respective time point; ^# p < 0.05 vs. time point 0. Percentage of changes in plasma acyl and desacyl ghrelin levels induced by LPS (C). Data are expressed as % change (mean ± sem) in the LPS group compared to vehicle at the respective time point. ** p < 0.01 vs. % change of desacyl ghrelin.

Fig. 2

LPS decreases plasma GOAT concentration, whereas protein content in the gastric corpus is slightly increased and mRNA expression unchanged in rats. Overnight fasted rats were injected ip with LPS (100μg/kg body weight) or vehicle (pyrogen-free saline) and trunk blood and stomach were collected at 2h post injection. Equal amounts of protein were loaded and plasma and gastric corpus mucosa GOAT concentrations were assessed using Western blot followed by semi-quantitative analysis. Lane 1 contains the molecular weight standards, lane 2 plasma proteins after vehicle injection, lane 3 plasma proteins after LPS, lane 4 gastric corpus mucosa proteins after vehicle and lane 5 gastric corpus mucosa proteins after LPS (A). The blot shows two dominant bands at ~50 and ~100 kDa. The 50 kDa band represents monomeric GOAT, whereas the 100 kDa band likely represents an SDS-stable dimer. Injection of LPS reduced the 50 kDa band (arrow) compared to vehicle demonstrating reduced plasma concentration of GOAT, whereas GOAT in the gastric corpus mucosa was increased (arrowhead, A). Re-staining of the Western blot with β-actin demonstrates equal gastric corpus mucosal protein concentration (A, insert). Quantification of GOAT plasma and stomach protein expression is shown in (B). Gastric GOAT mRNA expression did not change at 2h post LPS injection compared to vehicle treated rats (C). * p < 0.05 and *** p < 0.001 vs. vehicle; LPS, lipopolysaccharide; M, standard molecular weight marker; V, vehicle.

Fig. 3

LPS increases rectal temperature in rats. LPS (100μg/kg body weight) or vehicle was injected ip during the light phase in overnight fasted rats and rectal temperature measured in conscious lightly hand-restrained rats before and 2h, 5h, 7h and 24h post injection (A). Each line represents the mean ± sem of 5 rats/group. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. vehicle. Correlations between plasma acyl (B) or desacyl ghrelin (C) and rectal temperature from 0 - 24h post LPS injection. Values for r and p are indicated in each graph.

Fig. 4

LPS increases blood glucose levels in rats. Overnight fasted rats were injected ip with LPS (100μg/kg body weight) or vehicle (saline) and blood was withdrawn at the time points indicated at the x-axis for measurement of acyl and total ghrelin levels. At the same time, blood glucose levels were assessed (A). Each line represents the mean ± sem of 5 rats/group. * p < 0.05 and ** p < 0.01 vs. vehicle. Correlations between plasma acyl (B) or desacyl ghrelin (C) and blood glucose from 5h post LPS injection. Values for r and p are indicated in each graph.