Objective To explore the effect of ghrelin on insulin secretion and expression of glucose transporter protein-2 （Glut-2） in isolated pancreas of rats. Methods Twenty five Wistar rats were randomly devided into normal control group （NC group）， high concentration of glucose group （HCG group）， high concentration of glucose with high concentration of ghrelin group （10-8mol/L， HCG＋HCGh group）， medium concentration of ghrelin group（10-9mol/L， HCG＋MCGh group）， and low concentration of ghrelin group （10-10mol/L， HCG＋LCGh group） with 5 rats in each group. The rat isolated pancreas perfusion models were established firstly， then from the distal end of abdominal aortas， the models were perfused with low concentration of glucose （5.5mmol/L）， high concentration of glucose （33.3mmol/L） or high concentration of glucose added with different concentrations of ghrelin. Levels of insulin outflowed from portal vein were tested by ELISA method， expression levels of Glut-2 protein were tested by immunohistochemical method，and ultrastructure changes of islet β cell were observed under the transmission electron microscope. Results There were no significant difference on levels of fasting blood glucose （FBG）， fasting insulins （FINS）， homeostasis model of assess-ment for insulin resistence index （HOMA-IR）， and homeostasis model of assessment for pancreatic β cell function （HOMA-β），（P＞0.05）. There were no significant difference on insulin levels of effluent from portal vein of 5 groups （P＞0.05） when isolated pancreas perfused with 5.5mmol/L glucose， while had 2 secretion peaks in 3min and 10-12min after 33.3 mmol/L glucose perfusion， where HCG＋HCGh group at the top. The mean density value of Glut-2 protein in NC group was higher than that of other 4 groups （P＜0.05）. The results of transmission electron microscopy showed that apoptosis was lighter in NC group than that of other 4 groups， and apoptosis of HCG＋HCGh group was lighter than that of HCG＋MCGh group and HCG＋LCGh group. Conclusions In isolated pancreas of rats， ghrelin promotes high concentration of glucose-stimulated insulin secretion， decreases expression of Glut-2 protein， and protects the islet β cell.
ObjectiveTo study the effects of Ghrelin for glucose metabolism and insulin sensitivity of L6 rat myoblasts in palmitic acid induced, and to explore its possible mechanisms. MethodsThe L6 rat myoblasts were cultured until differentiation, then using palmitic acid(0.3 mmol/L) for 16 hours. The experimental group was treated with different doses of Ghrelin(1, 10, and 100 nmol/L) for 8 hours, then the glucose uptake was detected by using glucose oxidase peroxidase method(GOD-POD), the cell membrane glucose transporter 4(GLUT-4) protein staining was observated under confocal microscopy, and the expressions of total protein kinase B(Akt), phosphorylated protein kinase B(pAkt), total glycogen synthase kinase-3β(GSK-3β), and phosphorylated glycogen synthase kinase-3β(pGSK-3β) were detected by using immunoblotting(Western blot). ResultsGhrelin enhanced the glucose uptake of L6 rat myoblasts with insulin resistance, the cell membrane Glut-4 stain was deepen, the expressions of pAkt and pGSK-3βprotein increased, and this effect could be PI3K blocker(LY294002) eliminated. ConclusionGhrelin promotes the glucose uptake of L6 rat myoblasts through PI3K/Akt/GSK-3βsignaling pathway, so as to improve the sensitivity of insulin in L6 rats muscle cells.
ObjectiveTo explore the effect of exogenous ghrelin on early recovery of rats after subtotal gastrectomy. MethodsTwelve rats undergoing subtotal gastrectomy (B-Ⅰtype) were randomly divided into two groups, and saline or ghrelin was intraperitoneally injected in two groups, respectively. The body weight and daily food intake were measured before operation and on 1-7 d after operation. Rats were killed on day 7 after operation and the expressions of ghrelin mRNA in the fundus of stomach and anastomotic stoma was determined by realtime fluorescent quantitative PCR assay. The anastomotic bursting pressure and hydroxyproline content of anastomotic stoma tissues were also detected. ResultsThere was no significant difference (P＞0.05) in pre and postoperative body weight between two groups. Gradual decrease in postoperative body weight among the rats of saline group was observed which was significantly lower than that before operation (Plt;0.01). Body weight reached it’s lowest on day 1 after operation (Plt;0.01), after which it gradually increased but was still lower than that before operation (Plt;0.01). The postoperative body weight of rats in ghrelin group gradually decreased too, and was also significantly lower than preoperative body weight (Plt;0.01), except for the day 1 after operation (P=0.693). It reached the lowest on day 4 after operation (Plt;0.01), then it gradually increased but was still lower than that before operation (Plt;0.05 or Plt;0.01). The cumulative food intake of rats in ghrelin group was （52.50±6.77） g, which was significantly higher than that in saline group 〔(45.67±7.47) g〕, Plt;0.05. On day 7 after operation, relative expression of ghrelin mRNA in the fundus of stomach of rats in ghrelin group was 0.08±0.04, which was significantly lower than that in saline group (0.22±0.07), Plt;0.01. Compared with saline group, ghrelin-treated rats displayed significantly higher bursting pressure 〔（155.83±6.62） mm Hg vs. （172.33±10.44） mm Hg, Plt;0.05〕 higher hydroxyproline content 〔 (0.43±0.05) μg/mg wet tissue vs. （0.50±0.29） μg/mg wet tissue, Plt;0.01〕 at the anastomotic stoma. ConclusionGhrelin may effectively promote the early recovery of rats after subtotal gastrectomy.
Objective To establish the heterologous recombinant embryonic stem cell （ES cell） with ghrelin receptor (GHS-R) gene deletion in order to study the function of the GHS-R gene. Methods PGK-neo cassette was replaced by TK-neo in X-pPNT agent. The target region was located in exon 1 and exon 2 of GHS-R. Two homologous arms were amplified from mouse ES cells genomic DNA and constructed into X-pPNT with SalⅠ/NotⅠand EcoRⅠ/BamHⅠ, respectively. ES cells were electrotransfected with the linearized targeting vector and screened with G418 and Gancyclovir. Finally, the positive ES cell clones were identified by PCR and sequencing. Results The X-pPNT-TK-neo vector was obtained. And two homologous arms were inserted correctly. Finally, 328 positive clones were obtained by G418 and Gancyclovir screening, and 3 clones were confirmed as GHS-R gene homologous recombination. Conclusion This study provides the necessary basis for the establishment of the GHS-R knock out mouse model and the further study on GHS-R gene function in vivo.
ObjectiveTo investigate the possible mechanism of the improvement of type 2 diabetes mellitus with insulin resistance of skeletal muscles after Roux-en-Y gastric bypass surgery (RYGB). MethodsThirty GK rats were randomly divided into GK-RYGB group, sham operation group (GK-SO group), and control group (GK-control group); in addition, 10 Wistar rats served as normal control group.On day 28, the animals were sacrificed.The ghrelin concen-tration and PI3Kp85α, Akt/PKB, and GLUT4 levels were measured by ELISA, Western blot, and real-time PCR me-thods, respectively. Results①Compared with the GK-SO group and GK-control group, the plasma ghrelin levels were significantly increased in the normal control group (P < 0.01) and GK-RYGB group (P < 0.01).②Compared with the GK-SO group and GK-control group, p-/t-PI3Kp85α, p-/t-Akt/PKB, and m-/t-GLUT4 proteins were significantly incre-ased in the normal control group (P < 0.01, P < 0.05, and P < 0.01, respectively) and GK-RYGB group (P < 0.01, P < 0.05, and P < 0.01, respectively).③Compared with the GK-SO group and GK-control group, PI3Kp85α, Akt, and GLUT4 mRNA were significantly increased in the normal control group (P < 0.01, P < 0.05, and P < 0.05, respectively) and GK-RYGB group (P < 0.01, P < 0.05, and P < 0.05, respectively). ConclusionRYGB could elevate the ghrelin level obviously and upregulate PI3Kp85α, Akt/PKB, and GLUT4 levels and thus improve the insulin resistance of skeletal muscles of rats with T2DM.
ObjectiveTo research the change and significance of Ghrelin and Visfatin in plasma after Roux-en-Y gastric bypass surgery (RYGB) in type-2 diabetes (T2DM) rats. MethodsThirty healthy Sprague Dawley (SD) rats (8 weeks) were divided into T2DM group (n=22) and blank control group (CSO group, n=8). Then rats of T2DM group were fed with high calorie and high sugar diet for 6 weeks, following by one dose of streptozotocin via intraperitioneal injection. Finally, there were 18 T2DM rats were successfully established. Then those 18 T2DM rats were divided into two groups:RYGB group (n=10) and sham operation group (DSO group, n=8). Rats of RYGB underwent RYGB, rats of DSO group and CSO group underwent sham operation. Levels of fasting serum glucose (FBG), fasting serum insulin (FINS), Ghrelin, and Visfatin of rats in 3 groups were detected by enzyme-linked immunoassay (EIA) before and 4 weeks after operation, and calculating the lee index and insulin sensitivity index (ISI). ResultsIn RYGB group, compared with before operation, the body weight, lee index, levels of FBG, FINS, and Visfatin decreased after 4 weeks after operation (P < 0.050), but level of ISI and Ghrelin increased (P < 0.050), while there was no significant difference in body weight, body length, lee index, ISI, levels of FBG, FINS, Ghrelin, and Visfatin in DSO and CSO group before and 4 weeks after operation (P > 0.050). In addition, there was statistical difference among the 3 groups in difference before and after operation of Ghrelin and Visfatin, the difference before and after operation of Ghrelin and Visfatin was larger than those of DSO group and CSO group (P < 0.050), but the difference was not significant differed between DSO group and CSO group (P > 0.050). ConclusionsThe increase of plasma Ghrelin and the decrease of Visfatin play important role in the mechanism after RYGB in treatment of T2DM rats.