Weizhen Zhang

Ghrelin stimulates neurogenesis in the dorsal motor nucleus of the vagus

Ghrelin, a gastric peptide hormone, has been reported to regulate growth hormone secretion and energy homeostasis. Here we show that ghrelin promotes neural proliferation in vivo and in vitro in the rat dorsal motor nucleus of the vagus (DMNV). Ghrelin receptor mRNA and immunoreactivity were detected in tissues from DMNV. Systemic administration of ghrelin (130 nmol kg−1) significantly increased 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation in the DMNV in adult rats with cervical vagotomy (BrdU positive cells; from 27 ± 4 to 69 ± 14 n= 5, P < 0.05). In vitro, exposure of cultured DMNV neurones to ghrelin significantly increased the percentage of BrdU incorporation into cells in both dose‐dependent (10−9–10−6m), and time‐dependent (6 h to 48 h) manners. Ghrelin significantly increased voltage‐activated calcium currents in isolated single DMNV neurones from a mean maximal change of 141 ± 26 pA to 227 ± 37 pA. Upon removal of ghrelin, calcium currents slowly returned to baseline. Blocking L‐type calcium channels by diltiazem (10 μm) significantly attenuated ghrelin‐mediated increments in BrdU incorporation (n= 5, P < 0.05). Ghrelin acts directly on DMNV neurones to stimulate neurogenesis.

Effect of Des-acyl Ghrelin on Adiposity and Glucose Metabolism

Ghrelin, a gastric peptide hormone, has been reported to regulate GH secretion and energy homeostasis. Here, we examined the effect of des-acyl ghrelin driven from the fatty acid-binding protein-4 (FABP4) promoter on adiposity and glucose metabolism. A high level of expression of des-acyl ghrelin (692 +/- 293 fmol/g fat) in adipose tissue was detected in FABP4-ghrelin transgenic mice, but not in wild-type littermates. Circulating des-acyl ghrelin was significantly higher in FABP4-ghrelin transgenic mice (8409 +/- 3390 pm) compared with wild-type mice (513 +/- 58 pm). No significant change was observed for plasma acylated ghrelin and obestatin. Epididymal and perirenal fat masses decreased 35 +/- 9 and 52 +/- 9%, respectively, in FABP4-ghrelin transgenic mice. FABP4-ghrelin transgenic mice are resistant to obesity induced by high-fat diet. Brown fat mass was not affected by overexpression of ghrelin in adipose tissue. Glucose tolerance tests showed glucose levels to be significantly lower in FABP4-ghrelin transgenic mice than in controls after glucose administration. Insulin sensitivity testing showed that FABP4-ghrelin transgenic mice had a 28 +/- 5% greater hypoglycemic response to insulin. Our study demonstrates that overexpression of ghrelin from the FABP4 promoter impairs the development of white adipose tissues, and alters glucose tolerance and insulin sensitivity in mice.

Inhibition of pancreatic protein secretion by ghrelin in the rat

1 The role of ghrelin in the regulation of pancreatic protein secretion was investigated in vivo using anaesthetized rats with pancreatic ductal cannulas, and in isolated pancreatic acinar cells and pancreatic lobules in vitro. 2 In vivo, pancreatic protein output stimulated by CCK‐8 (400 pmol kg−1 h−1) was dose‐dependently inhibited by continuous ghrelin infusion (1.2 and 12 nmol kg−1 h−1) by 45 ± 8 and 84 ± 7 %, respectively. 3 In rats with acute subdiaphragmatic vagotomy, ghrelin (12 nmol kg−1 h−1) significantly inhibited CCK‐stimulated pancreatic protein secretion by 75 ± 18 %. 4 Infusion of ghrelin (12 nmol kg−1 h−1) abolished pancreatic protein secretion caused by the central vagal stimulant 2‐deoxy‐d‐glucose (75 mg kg−1), whereas bethanechol‐stimulated pancreatic protein output was inhibited by only 59 ± 7 %. 5 In vitro, ghrelin (10−11‐10−7m) produced no change in basal amylase release from dispersed, purified acinar cells. Co‐incubation of ghrelin (10−11−10−7m) with CCK−8 (10−10m) demonstrated no inhibition of CCK‐stimulated amylase release from dispersed acini. In contrast, ghrelin (10−9−10−7m) dose‐dependently inhibited amylase release from pancreatic lobules exposed to 75 mm potassium. 6 Our results show that (1) ghrelin is a potent inhibitor of pancreatic exocrine secretion in anaesthetized rats in vivo and in pancreatic lobules in vitro; and (2) the actions of ghrelin are indirect and may be exerted at the level of intrapancreatic neurons.

Gastric Mammalian Target of Rapamycin Signaling Regulates Ghrelin Production and Food Intake

Ghrelin, a gastric hormone, provides a hunger signal to the central nervous system to stimulate food intake. Mammalian target of rapamycin (mTOR) is an intracellular fuel sensor critical for cellular energy homeostasis. Here we showed the reciprocal relationship of gastric mTOR signaling and ghrelin during changes in energy status. mTOR activity was down-regulated, whereas gastric preproghrelin and circulating ghrelin were increased by fasting. In db/db mice, gastric mTOR signaling was enhanced, whereas gastric preproghrelin and circulating ghrelin were decreased. Inhibition of the gastric mTOR signaling by rapamycin stimulated the expression of gastric preproghrelin and ghrelin mRNA and increased plasma ghrelin in both wild-type and db/db mice. Activation of the gastric mTOR signaling by l-leucine decreased the expression of gastric preproghrelin and the level of plasma ghrelin. Overexpression of mTOR attenuated ghrelin promoter activity, whereas inhibition of mTOR activity by overexpression of TSC1 or TSC2 increased its activity. Ghrelin receptor antagonist d-Lys-3-GH-releasing peptide-6 abolished the rapamycin-induced increment in food intake despite that plasma ghrelin remained elevated. mTOR is therefore a gastric fuel sensor whose activity is linked to the regulation of energy intake through ghrelin.

Melanocortin-4 receptor-mediated inhibition of apoptosis in immortalized hypothalamic neurons via mitogen-activated protein kinase.

The melanocortin-4 receptor (MC4R) is a seven transmembrane member of the melanocortin receptor family. The GT1-1 cell line exhibits endogenous expression of MC4R. In this study, GT1-1 cells were used to study MC4R signaling pathways and to examine the effects of melanocortin receptor agonist NDP-MSH on apoptosis. MC4R mRNA expression was demonstrated by RT-PCR. Functional melanocortin receptor expression was implied by specific binding of NDP-MSH and cAMP production. NDP-MSH-stimulated GnRH release in a dose-dependent manner. Serum deprivation-induced apoptosis in GT1-1 cells, and the NDP-MSH inhibited this effect. The melanocortin receptor antagonist SHU9119 blocked the antiapoptotic actions of NDP-MSH, and the MAP kinase inhibitor PD98059 significantly attenuated the antiapoptotic effect. NDP-MSH-stimulated ERK1/2 phosphorylation in a dose-dependent manner. ERK1/2 phosphorylation could be abolished by SHU9119. In GT1-1 cells, melanocortin receptor activation causes ERK1/2 phosphorylation. In these cells, MC4R activation is also associated with antiapoptotic effects.

Intercellular Calcium Waves in Cultured Enteric Glia From Neonatal Guinea Pig

Enteric glia are important participants in information processing in the enteric nervous system. However, intercellular signaling mechanisms in enteric glia remain largely unknown. We postulated that intercellular calcium waves exist in enteric glia. Primary cultures of enteric glia were isolated from neonatal guinea pig taenia coli. Intracellular calcium in individual cells was quantified with fura‐2 AM microfluorimetry. Single‐cell stimulation was performed with a micromanipulator‐driven glass pipette. Data were expressed as mean ± SEM and analyzed by Students t‐test. Mechanical stimulation of a single enteric glial cell resulted in an increase in intracellular calcium, followed by concentric propagation to 36% ± 3% of neighboring cells. Intercellular calcium waves were blocked by depletion of intracellular calcium stores with thapsigargin (1 μM). Pretreatment of enteric glia with the phospholipase C inhibitor U73122 (1 μM) significantly decreased the percentage of cells responding to mechanical stimulation (6% ± 4%), but had no effect on waves induced by microinjection of the inositol trisphosphate (67% ± 13% vs. 60% ± 4% for control). Antagonism of inositol trisphosphate receptor attenuated intercellular calcium waves induced by both mechanical stimulation and microinjection of inositol trisphosphate. Uncoupling of gap junctions with octanol or heptanol significantly inhibited intercellular calcium wave propagation. Pretreatment of enteric glia with apyrase partially attenuated intercellular calcium waves. Our data demonstrate that enteric glial cells are capable of transmitting increases in intracellular calcium to surrounding cells, and that intercellular calcium waves involve a sequence of intracellular and extracellular steps in which phospholipase C, inositol trisphosphate, and ATP play roles. GLIA 42:252–262, 2003.

mTOR and the differentiation of mesenchymal stem cells

The mammalian target of rapamycin (mTOR), an evolutionarily conserved serine-threonine protein kinase, belongs to the phosphoinositide 3-kinase (PI3K)-related kinase family, which contains a lipid kinase-like domain within their C-terminal region. Recent studies have revealed that mTOR as a critical intracellular molecule can sense the extracellular energy status and regulate the cell growth and proliferation in a variety of cells and tissues. This review summarizes our current understanding about the effects of mTOR on cell differentiation and tissue development, with an emphasis on the lineage determination of mesenchymal stem cells. mTOR can promote adipogenesis in white adipocytes, brown adipocytes, and muscle satellite cells, while rapamycin inhibits the adipogenic function of mTOR. mTOR signaling may function to affect osteoblast proliferation and differentiation, however, rapamycin has been reported to either inhibit or promote osteogenesis. Although the precise mechanism remains unclear, mTOR is indispensable for myogenesis. Depending on the cell type, rapamycin has been reported to inhibit, promote, or have no effect on myogenesis.

Modulation of ghrelin O-acyltransferase expression in pancreatic islets.

Background: Ghrelin, the only identified circulating orexigenic signal, is unique in structure in which a specific acyl-modification of its third serine occurs. This acylation is necessary for ghrelin to bind to its receptor and to exert its biologic activity, which is catalyzed by ghrelin O-acyltransferase (GOAT). Although ghrelin is mainly secreted from gastric X/A like endocrine cells, it is also expressed in pancreatic islet cells and regulates insulin secretion. In this study, we examined the expression and regulation of GOAT in pancreas. Methods: GOAT mRNA and immunoreactivity were examined in pancreatic islets and INS-1 cells by RT-PCR and immunofluorescent staining or Western blotting. Results: Insulin inhibits the expression of GOAT mRNA and GOAT promoter activity in a dose and time-dependent manner. The mammalian target of rapamycin (mTOR) is activated by insulin. Blocking mTOR signaling by either rapamycin or overexpression of its negative regulator tuberous sclerosis complex 1 (TSC1) or TSC2 attenuates the inhibitory effect of insulin on the transcription and translation of GOAT. Conclusion: Our study suggests that GOAT is present in pancreatic islet cells and that insulin inhibits the expression of GOAT via the mediation of mTOR signaling.

The Growth Hormone Secretagogue Receptor: Its Intracellular Signaling and Regulation

The growth hormone secretagogue receptor (GHSR), also known as the ghrelin receptor, is involved in mediating a wide variety of biological effects of ghrelin, including: stimulation of growth hormone release, increase of food intake and body weight, modulation of glucose and lipid metabolism, regulation of gastrointestinal motility and secretion, protection of neuronal and cardiovascular cells, and regulation of immune function. Dependent on the tissues and cells, activation of GHSR may trigger a diversity of signaling mechanisms and subsequent distinct physiological responses. Distinct regulation of GHSR occurs at levels of transcription, receptor interaction and internalization. Here we review the current understanding on the intracellular signaling pathways of GHSR and its modulation. An overview of the molecular structure of GHSR is presented first, followed by the discussion on its signaling mechanisms. Finally, potential mechanisms regulating GHSR are reviewed.

Melanocortin-3 receptor activates MAP kinase via PI3 kinase.

HEK 293 cells stably expressing human melanocortin-3 receptor (MC3R) were exposed to melanocortin receptor agonist, NDP-MSH (10(-)(10)-10(-)(6) M). ERK1/2 was phosphorylated in a dose-dependent manner with an EC(50) of 3.3+/-1.5 x 10(-)(9) M, similar to the IC(50) of NDP-MSH binding to the MC3R. ERK1/2 phosphorylation was blocked by the melanocortin receptor antagonists SHU9119. NDP-MSH-induced ERK1/2 phosphorylation was sensitive to pertussis toxin and the PI3K inhibitor, wortmannin. Rp-cAMPS, BAPTA-AM and Myr-PKC did not inhibit the NDP-MSH-induced ERK1/2 phosphorylation. NDP-MSH stimulated cellular proliferation in a dose-dependent manner with a similar EC(50) to ERK1/2 phosphorylation, 2.1+/-0.6 x 10(-)(9) M. Cellular proliferation was blocked by AGRP (86-132) and by the MEK inhibitor, PD98059. The NDP-MSH did not inhibit serum deprivation-induced apoptosis. MC3R activation induces ERK1/2 phosphorylation via PI3K and this pathway is involved in cellular proliferation in HEK cells expressing MC3R.