Ghrelin, an orexigenic hormone released primarily from the gut, signals the hypothalamus to stimulate growth hormone release, enhance appetite and promote weight gain. The ghrelin receptor, aka Growth Hormone Secretagogue Receptor (GHS-R), is highly expressed in the brain, with highest expression in Agouti-Related Peptide (AgRP) neurons of the hypothalamus. We recently reported that neuron-specific deletion of GHS-R completely prevents diet-induced obesity (DIO) in mice by activating non-shivering thermogenesis. To further decipher the specific neuronal circuits mediating the metabolic effects of GHS-R, we generated AgRP neuron-specific GHS-R knockout mice (AgRP-Cre;Ghsr^f/f). Our data showed that GHS-R in AgRP neurons is required for ghrelin’s stimulatory effects on growth hormone secretion, acute food intake and adiposity, but not for long-term total food intake. Importantly, deletion of GHS-R in AgRP neurons attenuated diet-induced obesity (DIO) and enhanced cold-resistance in mice fed high fat diet (HFD). The HFD-fed knockout mice showed increased energy expenditure, and exhibited enhanced thermogenic activation in both brown and subcutaneous fat; this implies that GHS-R suppression in AgRP neurons enhances sympathetic outflow. In summary, our results suggest that AgRP neurons are key site for GHS-R mediated thermogenesis, and demonstrate that GHS-R in AgRP neurons plays crucial roles in governing energy utilization and pathogenesis of DIO.

Growth hormone secretagogue receptor (GHS-R) is widely known to regulate food intake and adiposity, but its role in glucose homeostasis is unclear. In this study, we investigated the expression of GHS-R in mouse pancreatic islets and its role in glycemic regulation. We used Ghsr-IRES-tauGFP mice, with Green Fluorescent Protein (GFP) as a surrogate for GHS-R, to demonstrate the GFP co-localization with insulin and glucagon expression in pancreatic islets, confirming GHS-R expression in β and α cells. We then generated β-cell-specific GHSR-deleted mice with MIP-Cre/ERT and validated that GHS-R suppression was restricted to the pancreatic islets. MIP-Cre/ERT;Ghsr^f/f mice showed normal energy homeostasis with similar body weight, body composition, and indirect calorimetry profile. Interestingly, MIP-Cre/ERT;Ghsr^f/f mice exhibited an impressive phenotype in glucose homeostasis. Compared to controls, MIP-Cre/ERT;Ghsr^f/f mice showed lower fasting blood glucose and insulin; reduced first-phase insulin secretion during a glucose tolerance test (GTT) and glucose-stimulated insulin secretion (GSIS) test in vivo. The isolated pancreatic islets of MIP-Cre/ERT;Ghsr^f/f mice also showed reduced insulin secretion during GSIS ex vivo. Further, MIP-Cre/ERT;Ghsr^f/f mice exhibited improved insulin sensitivity during insulin tolerance tests (ITT). Overall, our results confirmed GHS-R expression in pancreatic β and α cells; GHS-R cell-autonomously regulated GSIS and modulated systemic insulin sensitivity. In conclusion, β cell GHS-R was an important regulator of glucose homeostasis, and GHS-R antagonists may have therapeutic potential for Type 2 Diabetes.

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.

Ghrelin via its receptor, the growth hormone secretagogue receptor (GHS-R), increases food intake and adiposity. The tissue-specific functions of GHS-R in peripheral tissues are mostly unknown. We previously reported that while GHS-R expression is very low in white and brown fat of young mice, expression increases during aging. To investigate whether GHS-R has cell-autonomous effects in adipose tissues, we generated aP2-Cre-mediated GHS-R knockdown mice (aP2-Cre/Ghsr^f/f). We studied young (5–6 months) and old (15–17 months) aP2-Cre/Ghsr^f/f mice and their age-matched controls. Interestingly, young aP2-Cre/Ghsr^f/f mice had normal body weight but reduced fat; old mice showed pronounced reductions of both body weight and body fat. Calorimetry analysis revealed that aP2-Cre/Ghsr^f/f mice had normal food intake and locomotor activity at both young and old age; but intriguingly, while energy expenditure was normal at young age, it was significantly increased at old age. Both young and old aP2-Cre/Ghsr^f/f mice exhibited improved insulin sensitivity and glucose tolerance. Importantly, old aP2-Cre/Ghsr^f/f mice maintained higher core body temperature at 4 °C, and showed higher expression of the thermogenic uncoupling protein 1 (UCP1) gene. The ex vivo studies further demonstrated that GHS-R deficient white adipocytes from old mice exhibit increased glucose uptake and lipolysis, promoting lipid mobilization. Despite the fact that the in vivo phenotypes of aP2-Cre/Ghsr^f/f mice may not be exclusively determined by GHS-R knockdown in adipose tissues, our data support that GHS-R has cell-autonomous effects in adipocytes. The anabolic effect of GHS-R in adipocytes is more pronounced in aging, which likely contributes to age-associated obesity and insulin resistance.

The orexigenic hormone ghrelin increases food intake and promotes obesity through its receptor, growth hormone secretagogue receptor (GHS-R). We previously reported two neuron-specific GHS-R knockout mouse lines, namely pan-neuronal deletion by Syn1-cre and hypothalamic deletion by AgRP-cre, exhibiting differential diet-dependent effects on body weight. GHS-R deficiency in neurons elicited less pronounced metabolic effects under regular diet (RD) than high fat diet (HFD). While there was no difference in total food intake of HFD in either mouse line, Syn1-cre; Ghsr^f/f mice showed much greater anti-obesity effect than that of AgRP-cre; Ghsr^f/f mice. Meal feeding pattern is known to have a major impact on energy homeostasis and obesity development. Here, we investigated the feeding behaviors of these two neuron-specific GHS-R knockout mice under RD and HFD feeding, by assessing meal number, meal size, meal duration, and feeding frequency. Under the normal diet, RD-fed Syn1-cre; Ghsr^f/f mice showed a decreased meal size in dark phase, while RD-fed AgRP-cre; Ghsr^f/f mice showed an increased meal duration in dark phase. Under the obesogenic diet, HFD-fed Syn1-cre; Ghsr^f/f mice displayed reduced meal numbers in light phase and increased feeding in both light and dark phases, whereas HFD-fed AgRP-cre; Ghsr^f/f mice showed a decreased meal duration in the light phase only. Consistently, the expression of neuropeptides (Neuropeptide Y and Orexin) was increased in the hypothalamus of RD-fed Syn1-cre; Ghsr^f/f mice, whereas the expression of cannabinoid receptor type 1 (CB1) was increased in the hypothalamus of HFD fed Syn1-cre; Ghsr^f/f mice. Overall, feeding pattern changes were more pronounced in Syn1-cre; Ghsr^f/f mice than that in AgRP-cre; Ghsr^f/f mice, and HFD elicited greater alteration than RD. While AgRP-cre; Ghsr^f/f mice consumed HFD meals faster during the day (showing shorter meal duration), Syn1-cre; Ghsr^f/f mice ate few HFD meals during the light phase and ate slowly throughout the day (showing longer meal duration in both phases). Our findings reveal that neuronal GHS-R regulates energy homeostasis by altering feeding patterns, and differentially modulates feeding patterns in a site- and diet-dependent manner. The distinctive data in these two mouse lines also suggest that eating slowly during the optimal feeding period (dark phase for mice) may be beneficial in combating obesity.