Pieter-Jan Verhulst

Bitter taste receptors and α-gustducin regulate the secretion of ghrelin with functional effects on food intake and gastric emptying

Ghrelin is a hunger hormone with gastroprokinetic properties but the factors controlling ghrelin secretion from the stomach are unknown. Bitter taste receptors (T2R) and the gustatory G proteins, α-gustducin (gust) and α-transducin, are expressed in the gut and are involved in the chemosensation of nutrients. This study aimed to investigate whether T2R-agonists affect (i) ghrelin release via α-gustducin and (ii) food intake and gastric emptying via the release of ghrelin. The mouse stomach contains two ghrelin cell populations: cells containing octanoyl and desoctanoyl ghrelin, which were colocalized with α-gustducin and α-transducin, and cells staining for desoctanoyl ghrelin. Gavage of T2R-agonists increased plasma octanoyl ghrelin levels in WT mice but the effect was partially blunted in gust−/− mice. Intragastric administration of T2R-agonists increased food intake during the first 30 min in WT but not in gust−/− and ghrelin receptor knockout mice. This increase was accompanied by an increase in the mRNA expression of agouti-related peptide in the hypothalamus of WT but not of gust−/− mice. The temporary increase in food intake was followed by a prolonged decrease (next 4 h), which correlated with an inhibition of gastric emptying. The delay in emptying, which was partially counteracted by ghrelin, was not mediated by cholecystokinin and GLP-1 but involved a direct inhibitory effect of T2R-agonists on gastric contractility. This study is unique in providing functional evidence that activation of bitter taste receptors stimulates ghrelin secretion. Modulation of endogenous ghrelin levels by tastants may provide novel therapeutic applications for the treatment of weight -and gastrointestinal motility disorders.

Ghrelin's second life: from appetite stimulator to glucose regulator.

Ghrelin, a 28 amino acid peptide hormone produced by the stomach, was the first orexigenic hormone to be discovered from the periphery. The octanoyl modification at Ser³, mediated by ghrelin O-acyltransferase (GOAT), is essential for ghrelins biological activity. Ghrelin stimulates food intake through binding to its receptor (GRLN-R) on neurons in the arcuate nucleus of the hypothalamus. Ghrelin is widely expressed throughout the body; accordingly, it is implicated in several other physiological functions, which include growth hormone release, gastric emptying, and body weight regulation. Ghrelin and GRLN-R expression are also found in the pancreas, suggesting a local physiological role. Accordingly, several recent studies now point towards an important role for ghrelin and its receptor in the regulation of blood glucose homeostasis, which is the main focus of this review. Several mechanisms of this regulation by ghrelin have been proposed, and one possibility is through the regulation of insulin secretion. Despite some controversy, most studies suggest that ghrelin exerts an inhibitory effect on insulin secretion, resulting in increased circulating glucose levels. Ghrelin may thus be a diabetogenic factor. Obesity-related type 2 diabetes has become an increasingly important health problem, almost reaching epidemic proportions in the world; therefore, antagonists of the ghrelin-GOAT signaling pathway, which will tackle both energy- and glucose homeostasis, may be considered as promising new therapies for this disease.

The role of alpha-gustducin, in the effect of T2R-agonists on the secretion and physiological effects of ghrelin.

concentrations were higher in the healed group suggesting that the activity of elastase in these wounds was under more stringent control than in those patients that did not heal. Discussion: These results suggest that the higher SLPI concentrations found in healing wounds are integral to the prevention of inappropriate elastase activity at wound sites and thus the prevention of chronic wound development. This work was funded by a grant from the Dunhill Medical Trust.

Su2029 Medium Conditioned With Conventionally-Activated M1 Macrophages Inhibits Survival of Mouse Interstitial Cells of Cajal in Primary Culture

Background: Application of the surfactant benzylkonium chloride (BAC) to the serosal surface of the intestine causes ablative neurodegeneration within two days. Modest neurogenesis within 4-8 weeks of ablation has been observed in this model with neurons derived from glial precursors. We hypothesized that non-ablative concentrations of BAC would spare potential neural precursors and allow a more robust neuroregeneration. Aims: To determine whether low concentration BAC causes neurodegeneration and subsequent neurogenesis and whether markers of neural progenitors are expressed following BAC treatment. Methods: In C57Bl/6 mice, following laparotomy, a 1x0.2cm piece of absorbent paper soaked in 0.0001% BAC was applied to the serosal surface of the jejunum for 5 min. Following various periods of recovery, fixed segments of the external muscle layers were immunostained for HuC/D (mature enteric neurons), S100β (enteric glia), doublecortin (DCX; immature neurons), and/or SOX2 (neural pluripotency transcription factor). Immunoreactive (IR) cells were enumerated as the number of cells per ganglion. In some mice, EdU (100mg/kg IP) was administered daily between 12-16d post-BAC before animals were euthanized 21d postBAC. Results: In mice two days after BAC treatment there was a significant reduction in the number of HuC/D-IR cells per ganglion in the region of treatment (27 ± 8) compared to an orally-adjacent region (61 ± 7; P<0.05). In mice 21 days after BAC treatment, the number of neurons per ganglion in the treated region (45 ± 5) were no longer significantly less than the orally-adjacent region (61 ± 6; P<0.05). There was no evidence for EdU incorporation in HuC/D-IR cells. Although DCX-IR was detected in the rostral migratory stream of the CNS, DCX-IR was rarely detected in the myenteric plexus. SOX2-IR was increased in HuC/ D-IR neurons at 21d post-BAC. SOX2-IR was detected in S100β-IR cells as early as 2d postBAC and S100β-IR was detected in a proportion of HuC/D-IR cells in the BAC-treated region between 2-14d post-BAC. Conclusion: Low concentration BAC caused moderate neurodegeneration in the myenteric plexus of the mouse jejunum. The number of myenteric neurons increased by 21d post-BAC. The pluripotency transcription factor SOX2, which in the CNS is capable of reprogramming astrocytes to mature neurons, is expressed in enteric glial cells early following BAC treatment and in a proportion of mature neurons by 21d post-BAC. These data support the concept of neuroregeneration of the enteric nervous system, and suggest that SOX2 expression in enteric glia may drive adult neurogenesis without cell proliferation.

Role of the Energy Sensor AMP-Activated Protein Kinase (AMPK) and Its Downstream Effector Uncoupling Protein 2 (UCP2) in the Orexigenic Effect of Endogenous Ghrelin

Background. Ghrelin released from the stomach, stimulates food intake through stimulation of neuropeptide Y (NPY)/agouti-related peptide (AgRP) neurons in the hypothalamus. Several studies proposed a pivotal role for the energy sensor AMPK and UCP2 in ghrelins effects on NPY/AgRP expression and food intake stimulation, although most of these studies focused on the effects of exogenous ghrelin. Aim. To investigate whether a rise in endogenous ghrelin levels is able to influence NPY/AgRP expression via activation of AMPK activity and UCP2 expression. Methods. Endogenous ghrelin levels were increased in wildtype (GHS-R+/+) and ghrelin receptor knockout (GHS-R-/-) mice by fasting (24h) or by induction of streptozotocin (STZ)-diabetes (15 days). Plasma octanoylated ghrelin levels were determined by radioimmunoassay. The mRNA expression of AgRP, NPY and UCP2 in the hypothalamus was measured by real-time PCR. Hypothalamic AMPK activity in tissue lysates was measured with an immunoprecipitation kinase assay. Results.Octanoylated ghrelin levels peaked (4.5fold, P<0.01) after 24h of fasting and declined thereafter due to a decrease (1.7-fold, P<0.01) in the mRNA expression of ghrelin-O-acyl transferase (GOAT). GHS-R+/+ mice showed a significant increase in AgRP mRNA (from 0.33±0.03 to 1.03±0.09; P<0.001) and NPY mRNA (from 0.54 ± 0.08 to 0.88 ± 0.05; P<0.01) expression after 24h-fasting. In GHS-R-/mice no significant increase in AgRP and NPY mRNA expression was observed. Fasting did not affect AMPK activity in both genotypes but increased UCP2 mRNA expression (GHS-R+/+: from 0.35±0.05 to 0.50±0.04; P<0.05 and GHS-R-/-: from 0.41±0.04 to 0.55±0.08; P=0.058). The hyperghrelinemia associated with the induction of STZ-diabetes was accompanied by a significant increase in the expression of NPY and AgRP in GHS-R+/+ mice compared to nondiabetic controls (AgRP: from 0.22±0.12 to 2.74±0.45; P<0.001 and NPY: from 0.73±0.06 to 2.23±0.64; P<0.05) but not in GHS-R-/mice. AMPK activity in the hypothalamus of GHSR+/+ mice after induction of diabetes (20.2±1.6 pmol/min/mg) was decreased (P<0.05) compared to non-diabetic littermates (16.3±1.3 pmol/min/mg) and there was no genotypic difference. Similarly, UCP2 mRNA levels were decreased after the induction of STZ-diabetes compared to control mice in both genotypes (GHS-R+/+: from 0.68±0.02 to 0.26±0.03; P<0.001 and GHS-R-/-: from 0.61±0.05 to 0.28±0.05; P<0.01). Conclusion. Increasing endogenous ghrelin levels by fasting and by induction of diabetes stimulates the expression of AgRP and NPY in the hypothalamus via interaction with the GHS-R. The change in AMPK activity and its downstream effector, UCP2, accompanying these changes in endogenous ghrelin levels occur independently from the GHS-R suggesting that AMPK and UCP2 do not play a major role in the orexigenic effect of endogenous ghrelin.