Role and mechanism of action of the ghrelin receptor family in the regulation of energy and glucose homeostasis in mouse models of diabetes

Diabetes has become an increasingly important health problem, almost reaching epidemic proportions in the world. Type 1 diabetes, which predominantly affects young children, results from the autoimmune destruction of insulin-producing beta-cells, whereas type 2 diabetes is closely associated with obesity and characterized by a decreased peripheral insulin sensitivity. Besides health education and in order to develop new therapies, more insight into the physiological mechanisms governing food intake, body weight and blood glucose regulation during diabetes is indispensable.The gastrointestinal tract is well-positioned to sense the supply of recently ingested nutrients and this information is conveyed to thecentral nervous system, the master regulator of whole body energy homeostasis. These signals comprise several humoral factors released from thegastrointestinal tract, including the orexigenic hormone ghrelin which signals through the growth hormone secretagogue receptor. Ghrelin has been discovered as the first orexigenic hormone of the periphery with gastroprokinetic effects and recent evidence also suggests a role for ghrelin and its receptor in the regulation of blood glucose homeostasis.The general aim of this thesis was therefore to further establish the physiological functions of the ghrelin receptor and its closely related family member GPR39 in the pathogenesis of diabetes in diabetic mouse models. A role for ghrelin in meal initiation was earlier confirmed by the fluctuations in plasma ghrelin levels which increase immediately before a meal and rapidly fall after food intake, following a 24h pattern reciprocal to that of insulin. In agreement with this, increased plasma ghrelin levels were observed during insulin deficiency in rodents with streptozotocin (STZ)-induced diabetes, also characterized by hyperphagia and an altered gastric emptying. The first part of this studyaimed to investigate the causal relationship between these three eventsby comparing changes in food intake, glucose and gastric emptying in ghrelin receptor knockout (GHS-R-/-) and wild-type (GHS-R+/+) mice with STZ-induced diabetes. STZ-induced diabetes increased plasmaghrelin levels to a similar extent in both genotypes, while the hyperphagia was more pronounced in GHS-R+/+ than in GHS-R-/- mice. Concomitantly, the increase in the mRNA expression of the orexigenic hypothalamic neuropeptides, NPY and AgRP, was less pronounced in diabetic GHS-R-/- thanin GHS-R+/+ mice while the decrease in the mRNA expression of the anorexigenic neuropeptide, POMC, was unaffected by the genotype. In both genotypes, gastric emptying was accelerated to a similar extent. The acceleration of gastric emptying occurred independently from ghrelin and was not the cause of the hyperphagia because the increase in food intake was already apparent before the onset of the acceleration of gastric emptying. Contractility studies with smooth muscle strip preparations from the fundus of diabetic GHS-R+/+ and GHS-R-/- mice revealed that nitrergic relaxations were reduced whereas contractions were enhanced due to an improved affinity of muscarinic and tachykinergic receptors. Our results thus suggest that diabetic hyperphagia is regulated by centralpathways in which the ghrelin signaling pathway plays a predominant role by affecting the expression of NPY and AgRP in the hypothalamus, rather than through peripheral mechanisms. A pivotal role for the energy sensor AMPK and its downstream effector UCP2 in ghrelin s stimulatory effects on NPY/AgRP expression and food intake stimulation has been suggested, mostly by investigating the effects of exogenously administered ghrelin. The second part of this thesis therefore aimed to investigate whether a rise in endogenous ghrelin levels is able to influence hypothalamic AMPK activity, UCP2 and NPY/AgRP expression through activation of GHS-R. An increase in endogenous ghrelin levels was established by fasting (24h) or by induction of streptozotocin(STZ)-diabetes in GHS-R+/+ and GHS-R-/- mice. A significant increase in AgRP and NPY mRNA expression was observed after fasting and after the induction of STZ-diabetes, whereas this increase could not be observed in GHS-R-/- mice. Fasting did not affect AMPK activity and increased UCP2 mRNA expression similarly in both genotypes. AMPK activity and UCP2 expression in GHS-R+/+ mice after induction of diabetes was decreased and there was no genotypic difference. Exogenous ghrelin administration tended to decrease hypothalamic AMPK activity. It can be concluded that increasing endogenous ghrelin levels by fasting or STZ-induced diabetes stimulates the expression of AgRP and NPY via interaction with the GHS-R. The changes in AMPK activity and UCP2 occur independently from the GHS-R suggesting that they do not play a major role in the orexigenic effect of endogenous ghrelin. Another member of the ghrelin receptor family is GPR39, which may function as a Zn2+ sensor. The downregulation of GPR39 mRNA in adipose tissue of obese type 2 diabetic patients already suggested that GPR39 maycontribute to the pathogenesis of the disease. In the third part of this thesis we therefore wanted to investigate the role of GPR39 in the regulation of energy balance and glucose homeostasis in wild-type (GPR39+/+) and GPR39 knockout mice (GPR39-/-) with obesity-related type 2 diabetes. GPR39 mRNA levels in adipose tissue of fasted GPR39+/+ mice fed a high-fat diet (HFD) for 30 weeks were reduced and correlated positively with blood glucose levels. Body weight, fat percentage and energy intake were increased in the HFD-group but did not differ between both genotypes.Within the HFD-group, blood glucose levels were lower in GPR39-/- than in GPR39+/+ mice despite significant reductions in prandial plasma insulin levels. The latter was not due to changes in β-cell hyperplasia as immunohistochemical staining for insulin of pancreata of mice ona HFD showed no genotypic differences. The lower blood glucose levels may involve alterations in insulin sensitivity as revealed by glucose tolerance tests and respiratory quotient measurements which showed a preference of obese GPR39-/- mice for the use of carbohydrates as metabolic fuel. GPR39-/- mice fed a HFD displayed increased plasma ghrelin concentrations which may contribute to the alterations in glucose homeostasis. Gastric emptying and intestinal Zn2+ absorption were also determined in this study, showing that these are not involved in the regulation of glucose homeostasis. In summary our results suggest that GPR39 plays a role in the pathogenesis of obesity related type 2 diabetes by affecting the release and sensitivity to insulin. In conclusion, the results of the current thesis support the hypothesis that the ghrelin receptor family is an important player in the regulation of food intake and/or blood glucose during diabetes. Both the ghrelin receptor as well as the GPR39 should be considered as therapeutic targets for the treatment of diabetes.status: publishe