RESUMO
Insulin secretion from pancreatic ß cells is a key pillar of glucose homeostasis, which is impaired under obesity and aging. Growth hormone secretagogue receptor (GHSR) is the receptor of nutrient-sensing hormone ghrelin. Previously, we showed that ß-cell GHSR regulated glucose-stimulated insulin secretion (GSIS) in young mice. In the current study, we further investigated the effects of GHSR on insulin secretion in male mice under diet-induced obesity (DIO) and streptozotocin (STZ)-induced ß-cell injury in aging. ß-cell-specific-Ghsr-deficient (Ghsr-ßKO) mice exhibited no glycemic phenotype under DIO but showed significantly improved ex vivo GSIS in aging. We also detected reduced insulin sensitivity and impaired insulin secretion during aging both in vivo and ex vivo. Accordingly, there were age-related alterations in expression of glucose transporter, insulin signaling pathway, and inflammatory genes. To further determine whether GHSR deficiency affected ß-cell susceptibility to acute injury, young, middle-aged, and old Ghsr-ßKO mice were subjected to STZ. We found that middle-aged and old Ghsr-ßKO mice were protected from STZ-induced hyperglycemia and impaired insulin secretion, correlated with increased expression of insulin signaling regulators but decreased pro-inflammatory cytokines in pancreatic islets. Collectively, our findings indicate that ß-cell GHSR has a major impact on insulin secretion in aging but not obesity, and GHSR deficiency protects against STZ-induced ß-cell injury in aging.
Assuntos
Envelhecimento , Células Secretoras de Insulina , Obesidade , Receptores de Grelina , Animais , Masculino , Camundongos , Glicemia/metabolismo , Diabetes Mellitus Experimental , Hiperglicemia , Insulina/metabolismo , Resistência à Insulina , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/metabolismo , Receptores de Grelina/metabolismo , Receptores de Grelina/genética , Transdução de Sinais , Estreptozocina/toxicidadeRESUMO
Ghrelin and obestatin are two "sibling proteins" encoded by the same preproghrelin gene but possess an array of diverse and complex functions. While there are ample literature documenting ghrelin's functions, the roles of obestatin are less clear and controversial. Ghrelin and obestatin have been perceived to be antagonistic initially; however, recent studies challenge this dogma. While they have opposing effects in some systems, they function synergistically in other systems, with many functions remaining debatable. In this review, we discuss their functional relationship under three "C" categories, namely complex, complementary, and contradictory. Their functions in food intake, weight regulation, hydration, gastrointestinal motility, inflammation, and insulin secretion are complex. Their functions in pancreatic beta cells, cardiovascular, muscle, neuroprotection, cancer, and digestive system are complementary. Their functions in white adipose tissue, thermogenesis, and sleep regulation are contradictory. Overall, this review accumulates the multifaceted functions of ghrelin and obestatin under both physiological and pathological conditions, with the intent of contributing to a better understanding of these two important gut hormones.
Assuntos
Tecido Adiposo Branco , Grelina , Tecido Adiposo Branco/metabolismo , Grelina/metabolismo , Secreção de InsulinaRESUMO
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.
Assuntos
Ilhotas Pancreáticas , Receptores de Grelina , Animais , Grelina/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Insulina/metabolismoRESUMO
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 ß and α cells. We then generated ß-cell-specific GHSR-deleted mice with MIP-Cre/ERT and validated that GHS-R suppression was restricted to the pancreatic islets. MIP-Cre/ERT;Ghsrf/f mice showed normal energy homeostasis with similar body weight, body composition, and indirect calorimetry profile. Interestingly, MIP-Cre/ERT;Ghsrf/f mice exhibited an impressive phenotype in glucose homeostasis. Compared to controls, MIP-Cre/ERT;Ghsrf/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;Ghsrf/f mice also showed reduced insulin secretion during GSIS ex vivo. Further, MIP-Cre/ERT;Ghsrf/f mice exhibited improved insulin sensitivity during insulin tolerance tests (ITT). Overall, our results confirmed GHS-R expression in pancreatic ß and α cells; GHS-R cell-autonomously regulated GSIS and modulated systemic insulin sensitivity. In conclusion, ß cell GHS-R was an important regulator of glucose homeostasis, and GHS-R antagonists may have therapeutic potential for Type 2 Diabetes.
Assuntos
Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Receptores de Grelina/metabolismo , Animais , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Camundongos , Camundongos Knockout , Receptores de Grelina/genéticaRESUMO
Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: α cells secrete glucagon, ß cells secrete insulin, δ cells secrete somatostatin, ε cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are ß cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250-350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.
Assuntos
Separação Celular/métodos , Ilhotas Pancreáticas/citologia , Animais , Colagenases , CamundongosRESUMO
BACKGROUND: Thermogenic impairment promotes obesity and insulin resistance. Adiponectin is an important regulator of energy homeostasis. While many beneficial metabolic effects of adiponectin resemble that of activated thermogenesis, the role of adiponectin in thermogenesis is not clear. In this study, we investigated the role of adiponectin in thermogenesis using adiponectin-null mice (Adipoq -/-). METHODS: Body composition was measured using EchoMRI. Metabolic parameters were determined by indirect calorimetry. Insulin sensitivity was evaluated by glucose- and insulin- tolerance tests. Core body temperature was measured by a TH-8 temperature monitoring system. Gene expression was assessed by real-time PCR and protein levels were analyzed by Western blotting and immunohistochemistry. The mitochondrial density of brown adipose tissue was quantified by calculating the ratio of mtDNA:total nuclear DNA. RESULTS: Under normal housing temperature of 24 °C and ad libitum feeding condition, the body weight, body composition, and metabolic profile of Adipoq -/- mice were unchanged. Under fasting condition, Adipoq -/- mice exhibited reduced energy expenditure. Conversely, under cold exposure, Adipoq -/- mice exhibited reduced body temperature, and the expression of thermogenic regulatory genes was significantly reduced in brown adipose tissue (BAT) and subcutaneous white adipose tissue (WAT). Moreover, we observed that mitochondrial content was reduced in BAT and subcutaneous WAT, and the expression of mitochondrial fusion genes was decreased in BAT of Adipoq -/- mice, suggesting that adiponectin ablation diminishes mitochondrial biogenesis and altered mitochondrial dynamics. Our study further revealed that adiponectin deletion suppresses adrenergic activation, and down-regulates ß3-adrenergic receptor, insulin signaling, and the AMPK-SIRT1 pathway in BAT. CONCLUSIONS: Our findings demonstrate that adiponectin is an essential regulator of thermogenesis, and adiponectin is required for maintaining body temperature under cold exposure.
Assuntos
Adiponectina/fisiologia , Temperatura Baixa , Termogênese , Adiponectina/genética , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Comportamento Animal , DNA Mitocondrial/metabolismo , Meio Ambiente , Jejum , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse FisiológicoRESUMO
Ghrelin increases food intake and body weight by stimulating orexigenic agouti-related protein (AgRP)/neuropeptide Y (NPY) neurons and inhibiting anorexic pro-opiomelanocortin (POMC) neurons in the hypothalamus. Growth hormone secretagogue receptor (Ghsr) mediates the effect of ghrelin on feeding behavior and energy homeostasis. However, the role of Ghsr in the ghrelin effect on these two populations of neurons is unclear. We hypothesized that Ghsr mediates the effect of ghrelin on AgRP and POMC neurons. In this study, we determined whether Ghsr similarly mediates the effects of ghrelin on AgRP/NPY and POMC neurons using cell type-specific Ghsr-knockout mice. Perforated whole-cell recordings were performed on green fluorescent protein-tagged AgRP/NPY and POMC neurons in the arcuate nucleus in hypothalamic slices. In Ghsr+/+ mice, ghrelin (100 nM) significantly increased the firing activity of AgRP/NPY neurons but inhibited the firing activity of POMC neurons. In Ghsr-/- mice, the excitatory effect of ghrelin on AgRP/NPY neurons was abolished. Ablation of Ghsr also eliminated ghrelin-induced increases in the frequency of GABAergic inhibitory postsynaptic currents of POMC neurons. Strikingly, ablation of Ghsr converted the ghrelin effect on POMC neurons from inhibition to excitation. Des-acylated ghrelin had no such effect on POMC neurons in Ghsr-/- mice. In both Ghsr+/+ and Ghsr-/- mice, blocking GABAA receptors with gabazine increased the basal firing activity of POMC neurons, and ghrelin further increased the firing activity of POMC neurons in the presence of gabazine. Our findings provide unequivocal evidence that Ghsr is essential for ghrelin-induced excitation of AgRP/NPY neurons. However, ghrelin excites POMC neurons through an unidentified mechanism that is distinct from conventional Ghsr.
Assuntos
Grelina/farmacologia , Hipotálamo/metabolismo , Neurônios/metabolismo , Neuropeptídeo Y/metabolismo , Receptores de Grelina/metabolismo , Proteína Relacionada com Agouti/metabolismo , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Ingestão de Alimentos/efeitos dos fármacos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp/métodos , Pró-Opiomelanocortina/metabolismoRESUMO
Orexigenic hormone ghrelin and anorexic hormone obestatin are encoded by the same preproghrelin gene. While it is known that ghrelin inhibits glucose-stimulated insulin secretion (GSIS), the effect of obestatin on GSIS is unclear. Ghrelin's effect is mediated by its receptor Growth Hormone Secretagogue Receptor (GHS-R), but the physiologically relevant receptor of obestatin remains debatable. Here we have investigated the effect of obestatin on GSIS in vitro, in vivo and ex vivo, and tested whether obestatin regulates insulin secretion through GHS-R. We found that under hyperglycemic condition, obestatin augments GSIS in rat insulinoma cells (INS-1) and in pancreatic islets from ghrelin -/- mice. Surprisingly, obestatin-induced GSIS was absent in ß-cells in which GHS-R was suppressed. Obestatin-induced insulin secretion was abolished in the circulation of Ghsr -/- mice, and in pancreatic islets isolated from Ghsr -/- mice. We also found that obestatin-induced GSIS was attenuated in islets isolated from ß-cell-specific Ghsr knockout MIP-Cre/ERT;Ghsrf/f mice. Our data collectively demonstrate that obestatin is a potent insulin secretagogue under hyperglycemic condition, and obestatin's effect on insulin secretion is mediated by GHS-R in pancreatic ß-cells. Our findings reveal an intriguing insight that obestatin and ghrelin have opposing effects on insulin secretion, and both are mediated through ghrelin receptor GHS-R.
Assuntos
Grelina/genética , Glucose/farmacologia , Insulina/metabolismo , Receptores de Grelina/metabolismo , Animais , Células Cultivadas , Técnicas de Inativação de Genes , Grelina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , RatosRESUMO
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;Ghsrf/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.
Assuntos
Proteína Relacionada com Agouti/metabolismo , Neurônios/metabolismo , Obesidade/etiologia , Obesidade/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores de Grelina/genética , Receptores de Grelina/metabolismo , Termogênese , Animais , Dieta Hiperlipídica , Modelos Animais de Doenças , Metabolismo Energético , Comportamento Alimentar , Deleção de Genes , Hormônio do Crescimento/metabolismo , Homeostase , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Knockout , Modelos BiológicosRESUMO
Ghrelin signaling has major effects on energy and glucose homeostasis, but it is unknown whether ghrelin's functions are centrally and/or peripherally mediated. The ghrelin receptor, growth hormone secretagogue receptor (GHS-R), is highly expressed in the brain and detectable in some peripheral tissues. To understand the roles of neuronal GHS-R, we generated a mouse line where Ghsr gene is deleted in all neurons using synapsin 1 (Syn1)-Cre driver. Our data showed that neuronal Ghsr deletion abolishes ghrelin-induced spontaneous food intake but has no effect on total energy intake. Remarkably, neuronal Ghsr deletion almost completely prevented diet-induced obesity (DIO) and significantly improved insulin sensitivity. The neuronal Ghsr-deleted mice also showed improved metabolic flexibility, indicative of better adaption to different fuels. In addition, gene expression analysis suggested that hypothalamus and/or midbrain might be the sites that mediate the effects of GHS-R in thermogenesis and physical activity, respectively. Collectively, our results indicate that neuronal GHS-R is a crucial regulator of energy metabolism and a key mediator of DIO. Neuronal Ghsr deletion protects against DIO by regulating energy expenditure, not by energy intake. These novel findings suggest that suppressing central ghrelin signaling may serve as a unique antiobesity strategy.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Neurônios/metabolismo , Obesidade/metabolismo , Obesidade/prevenção & controle , Receptores de Grelina/metabolismo , Animais , Encéfalo/metabolismo , Calorimetria Indireta , Ingestão de Alimentos/genética , Ingestão de Alimentos/fisiologia , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Feminino , Teste de Tolerância a Glucose , Hipotálamo/metabolismo , Resistência à Insulina/genética , Resistência à Insulina/fisiologia , Masculino , Camundongos , Camundongos Mutantes , Obesidade/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Grelina/genética , Sinapsinas/genética , Sinapsinas/metabolismo , Termogênese/genética , Termogênese/fisiologiaRESUMO
PURPOSE OF REVIEW: Ghrelin is a multifaceted gut hormone that activates its receptor, growth hormone secretagogue receptor (GHS-R). Ghrelin's hallmark functions are its stimulatory effects on growth hormone release, food intake and fat deposition. Ghrelin is famously known as the 'hunger hormone'. However, ample recent literature indicates that the functions of ghrelin go well beyond its role as an orexigenic signal. Here, we have reviewed some of the most recent findings on ghrelin and its signalling in animals and humans. RECENT FINDINGS: Ghrelin regulates glucose homeostasis by inhibiting insulin secretion and regulating gluconeogenesis/glycogenolysis. Ghrelin signalling decreases thermogenesis to regulate energy expenditure. Ghrelin improves the survival prognosis of myocardial infarction by reducing sympathetic nerve activity. Ghrelin prevents muscle atrophy by inducing muscle differentiation and fusion. Ghrelin regulates bone formation and metabolism by modulating proliferation and differentiation of osteoblasts. SUMMARY: In addition to ghrelin's effects on appetite and adiposity, ghrelin signalling also plays crucial roles in glucose and energy homeostasis, cardioprotection, muscle atrophy and bone metabolism. These multifaceted roles of ghrelin make ghrelin and GHS-R highly attractive targets for drug development. Ghrelin mimetics may be used to treat heart diseases, muscular dystrophy/sarcopenia and osteoporosis; GHS-R antagonists may be used to treat obesity and insulin resistance.