Peptide dual agonists toward both glucagon-like peptide 1 receptor (GLP-1R) and glucagon receptor (GCGR) are emerging as novel therapeutics for the treatment of type 2 diabetes mellitus (T2DM) patients with obesity. Our previous work identified a Xenopus GLP-1-based dual GLP-1R/GCGR agonist termed xGLP/GCG-13, which showed decent hypoglycemic and body weight lowering activity. However, the clinical utility of xGLP/GCG-13 is limited due to its short in vivo half-life. Inspired by the fact that O-GlcNAcylation of intracellular proteins leads to increased stability of secreted proteins, we rationally designed a panel of O-GlcNAcylated xGLP/GCG-13 analogs as potential long-acting GLP-1R/ GCGR dual agonists. One of the synthesized glycopeptides 1f was found to be equipotent to xGLP/GCG-13 in cell-based receptor activation assays. As expected, O-GlcNAcylation effectively improved the stability of xGLP/GCG-13 in vivo. Importantly, chronic administration of 1f potently induced body weight loss and hypoglycemic effects, improved glucose tolerance, and normalized lipid metabolism and adiposity in both db/db and diet induced obesity (DIO) mice models. These results supported the hypothesis that glycosylation is a useful strategy for improving the in vivo stability of GLP-1-based peptides and promoted the development of dual GLP-1R/GCGR agonists as antidiabetic/antiobesity drugs.
Mice ; Animals ; Glucagon-Like Peptide 1/metabolism* ; Receptors, Glucagon/therapeutic use* ; Xenopus laevis/metabolism* ; Diabetes Mellitus, Type 2/drug therapy* ; Glycopeptides/therapeutic use* ; Obesity/drug therapy* ; Hypoglycemic Agents/pharmacology* ; Peptides/pharmacology*

OBJECTIVE: To study the effect of exendin-4(Ex-4) on the differentiation of neural stem cells(NSCs) in adult mouse subventricular zone(SVZ)and its mechanism . METHODS: NSCs in the SVZ were derived from 5-week C57BL/6J mice and the expression of nestin was detected by immunofluorescence. The cell morphology was observed after the cells treatmed with 100 nmol/L Ex-4 for 14 days.The expressions of nestin and glucagon-like peptide-1 receptor (GLP-1R) were detected by immunofluorescence. GLP-1R was knocked down by using shRNA and the study was divided into four groups: control group, Ex-4 group, GLP-1R knockdown group, GLP-1R knockdown + Ex-4 group. After treatment with 100 nmol/L Ex-4 for 14 d, β-tublin III and glial fibrillary acidic protein (GFAP) were labeled by immunofluorescence and then the proportion of β-tublin III positive cells were counted. Western blot was used to detect the activation of cAMP-response element binding protein (CREB) in NSCs. In order to further study the effects of Ex-4 on mitogen-activated protein kinase(MAPK) and phosphatidylinositol 3-hydroxy kinase (PI3K) pathways, the cells were pretreated with MAPK inhibitor U0126 at a concentration of 0.07 μmol/L for 30 min or PI3K inhibitor LY294002 at 50 μmol for 2 h, respectively. The study was divided into six groups: control group, Ex-4 group, U0126 group, U0126 + Ex-4 group, LY294002 group, LY294002 + Ex-4 group. The activation of CREB in each group was detected by Western blot. The experiment was repeated three times independently. RESULTS: NSCs were successfully extracted from SVZ of C57BL/6J mice. Immunofluorescence showed that nestin and GLP-1R were positive in NSCs. Compared with the control group, the proportion of neurons differentiated from Ex-4 group was higher. The percentage of neurons in GLP-1R knockdown + Ex-4 group was basically the same as that in control group (P＜0.01). The positive cells of beta-tublin III showed positive activation of GLP-1R and CREB. Western blot showed that CREB was significantly activated in the Ex-4 group, and knockdown of GLP-1R abolished its activation (P＜0.01). U0126 did not affect Ex-4-mediated CERB activation, and LY294002 significantly reduced Ex-4-mediated CREB activation (P＜0.01). CONCLUSION Ex-4 promotes the differentiation of NSCs into neurons in SVZ of adult mice through GLP-1R receptor, which may be achieved through PI3K/CREB pathway.
Animals ; Cell Differentiation ; Cells, Cultured ; Cyclic AMP Response Element-Binding Protein ; metabolism ; Exenatide ; pharmacology ; Gene Knockdown Techniques ; Glucagon-Like Peptide-1 Receptor ; genetics ; metabolism ; Lateral Ventricles ; cytology ; Mice ; Mice, Inbred C57BL ; Neural Stem Cells ; cytology ; Phosphatidylinositol 3-Kinases

Glucagon-like peptide-1 (GLP-1) expression is shared by both intestinal cells and neurons of brainstem, which plays anorexigenic role on food intake. However, the exact source of physiological GLP-1 influencing food intake and pertinent mechanism of GLP-1 receptor agonists (GLP-1RA) remain unelucidated. In this study, the immediate early gene product c-Fos was chosen as the specific antigen for immunohistochemistry to show the certain areas of central nervous system (CNS) activation by the GLP-1RA. Thirty normal SD rats were randomly assigned to 3 groups, which were single intraperitoneally injected with Liraglutide (200 μg/kg), Exenatide (10 μg/kg) and saline, respectively. After injection, the amount of food intake and acute glycemic variation were assessed for comparison. The results showed that acute pharmacological dosage of GLP-1RA (Liraglutide or Exenatide) could significantly influence food intake. However, glycemic change indicated that the anorexic effect was dissociated with change in blood glucose in normal rats. Moreover, c-Fos was expressed significantly higher in major critical nuclei related to food intake in GLP-1RA groups when compared with the control group, and its expression was also found in spinal cord. The results suggested that acute administration of pharmacological doses of GLP-1 influences CNS via circulation and vagal pathways, especially on the arcuate nucleus (ARC) and the nucleus of solitary tract (NTS), and GLP-1 modulates autonomic nervous activities.
Animals ; Eating ; drug effects ; Exenatide ; pharmacology ; Glucagon-Like Peptide-1 Receptor ; agonists ; Liraglutide ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley

To investigate the effect of berberine on glycemia regulation in rats with diabetes and the related mechanisms.Diabetic-like rat model was successfully induced by intraperitoneal injection of streptozotocin in 50 out of 60 male SD rats, which were then randomly divided into 5 groups with 10 rats in each:control group (received vehicle only), positive drug control group (sitagliptin 10 mg·kg·d), low-dose berberine group (30 mg·kg·d), moderate-dose berberine group (60 mg·kg·d), and high-dose berberine group (120 mg·kg·d). All animals were fed for 3 d, and fasting blood sampling was performed on day 3 of administration. Rats were given glucose (2 g/kg) by gavage 30 min after the last dose. Blood and intestinal samples were obtained 2 h after glucose loading. Fasting blood glucose (FBG) and 2-h postprandial plasma glucose (2h-PPG) were detected by using biochemical analyzer, and insulin, glucagon-like peptide-1 (GLP-1) and dipeptidyl peptidase-Ⅳ(DPP-Ⅳ) were measured by using ELISA kit.No significant difference in FBG and serum DPP-Ⅳ level were found between berberine groups and control group (all>0.05). Compared with control group, serum levels of GLP-1 and insulin were increased in high-and moderate-dose berberine groups, while 2h-PPG was decreased (all<0.05); GLP-1 levels in the intestinal samples were increased, while DPP-Ⅳ levels were decreased in all berberine groups (all<0.05).Short-term berberine administration can decrease 2h-PPG level in streptozotocin-induced diabetic rat model through local inhibition of intestinal DPP-Ⅳ. The efficacy of DPP-Ⅳ inhibitor may be associated with its intestinal pharmacokinetics.
Animals ; Berberine ; pharmacokinetics ; pharmacology ; Blood Glucose ; drug effects ; Diabetes Mellitus, Experimental ; chemically induced ; drug therapy ; Dipeptidyl Peptidase 4 ; analysis ; drug effects ; pharmacokinetics ; Dipeptidyl-Peptidase IV Inhibitors ; Dose-Response Relationship, Drug ; Glucagon-Like Peptide 1 ; analysis ; blood ; Hypoglycemic Agents ; Insulin ; blood ; Intestines ; chemistry ; drug effects ; Male ; Rats ; Rats, Sprague-Dawley ; Sitagliptin Phosphate

OBJECTIVETo investigate the role of p38MAPK signaling pathway in the mechanism by which glucagon-like peptide-1 (GLP-1) inhibits endothelial cell damage induced by AGEs.

METHODSHuman umbilical vein endothelial cells were divided into control group, AGEs group, GLP-1 group, AGEs+GLP-1 group, AGEs+inhibitor group, and AGEs+GLP-1+inhibitor group. The expressions of p-p38MAPK/p38MAPK and p-eNOS/eNOS protein were examined by Western blotting, and the cell apoptosis rates were tested by flow cytometry.

RESULTSCompared with the control group, AGEs significantly enhanced the expression of p-p38 MAPK protein (P=0.001) while GLP-1 significantly inhibited its expression (P<0.001). AGEs significantly inhibited the expression of p-eNOS protein (P=0.007), which was enhanced by GLP-1 and p38 MAPK inhibitor (SB203580) (P=0.004). Both SB203580 and GLP-1 treatment decreased the apoptosis rate of AGEs-treated cells (P<0.001).

CONCLUSIONGLP-1 can protect human umbilical vein endothelial cells against AGEs-induced apoptosis partially by inhibiting the phosphorylation of p38MAPK protein and promoting the expression of p-eNOS protein.

Apoptosis ; Cells, Cultured ; Glucagon-Like Peptide 1 ; pharmacology ; Glycation End Products, Advanced ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; metabolism ; Humans ; Nitric Oxide Synthase Type III ; metabolism ; Phosphorylation ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo investigate the effect of glucagon-like peptide 1 (GLP-1) on cognitive dysfunction in diabetic rats.

METHODSMale SD rats were randomly divided into normal control group, diabetes mellitus (DM) group, and GLP-1 treatment group. Rat models of type 2 diabetes were established by high-sugar and high-fat feeding and streptozotocin (STZ) injection, and 25 days after the onset of diabetes, GLP-1 was infused in GLP-1 treatment group at the rate of 30 pmol·kg·minvia a subcutaneous osmotic pump for 7 days. The learning and cognitive ability of the rats was assessed with Morris water maze test, and the expression of cognition-related genes in the hippocampus tissue was detected with real-time PCR, Western blotting and immunohistochemical staining.

RESULTSCompared with the normal control group, the diabetic rats showed significantly decreased learning and memory abilities (P<0.05) with increased hippocampal expressions of APP, BACE1, Arc, ERK1/2, PKA, and PKC mRNAs (P<0.05) and Arc protein. Compared with diabetic rats, GLP-1-treated rats showed significantly improvements in the learning and memory function (P<0.05) with decreased expressions of APP, BACE1, Arc, ERK1/2, and PKA mRNAs (P<0.05) and Arc protein.

CONCLUSIONGLP-1 can improve cognitive dysfunctions in diabetic rats possibly by regulating the PKC, PKA, and ERK1/2 pathways and inhibiting Arc expression in the hippocampus.

Animals ; Diabetes Mellitus, Experimental ; drug therapy ; Diabetes Mellitus, Type 2 ; drug therapy ; Glucagon-Like Peptide 1 ; pharmacology ; Hippocampus ; drug effects ; Learning ; drug effects ; Male ; Memory ; drug effects ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Streptozocin

OBJECTIVETo investigate the effects of activation of the GLP-1 receptor on the p38MAPK signaling pathway in hepatic stellate cells (HSCs).

METHODSHSCs were isolated and identified according to morphological features; the levels of GLP-1R protein were determined by western blotting.The HSCs were randomly divided into a control grouP (normal saline treatment) and experimental grouP(liraglutide treatment); after 120 hours, the expression of p38MAPK mRNA was examined by RT-PCR and of phosphorylated (p)-p38MAPK protein was detected by western blotting.

RESULTSGLP-1R proteins were detected in the HSCs. Compared with the control group, the experimental group showed significantly decreased p38MAPK mRNA and p-p38MAPK protein (both P < 0.01).

CONCLUSIONThe p38MAPK signaling pathway could be down-regulated when GLP-1R is activated in HSCs.

Cells, Cultured ; Glucagon-Like Peptide 1 ; analogs & derivatives ; pharmacology ; Glucagon-Like Peptide-1 Receptor ; Hepatic Stellate Cells ; metabolism ; Humans ; Liraglutide ; MAP Kinase Signaling System ; RNA, Messenger ; Receptors, Glucagon ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo reveal the effects and related mechanisms of chlorogenic acid (CGA) on intestinal glucose homeostasis.

METHODSForty male Sprague-Dawley rats were randomly and equally divided into four groups: normal chow (NC), high-fat diet (HFD), HFD with low-dose CGA (20 mg/kg, HFD-LC), and HFD with high-dose CGA (90 mg/kg, HFD-HC). The oral glucose tolerance test was performed, and fast serum insulin (FSI) was detected using an enzyme-linked immunosorbent assay. The mRNA expression levels of glucose transporters (Sglt-1 and Glut-2) and proglucagon (Plg) in different intestinal segments (the duodenum, jejunum, ileum, and colon) were analyzed using quantitative real-time polymerase chain reaction. SGLT-1 protein and the morphology of epithelial cells in the duodenum and jejunum was localized by using immunofluorescence.

RESULTSAt both doses, CGA ameliorated the HFD-induced body weight gain, maintained FSI, and increased postprandial 30-min glucagon-like peptide 1 secretion. High-dose CGA inhibited the HFD-induced elevation in Sglt-1 expression. Both CGA doses normalized the HFD-induced downregulation of Glut-2 and elevated the expression of Plg in all four intestinal segments.

CONCLUSIONAn HFD can cause a glucose metabolism disorder in the rat intestine and affect body glucose homeostasis. CGA can modify intestinal glucose metabolism by regulating the expression of intestinal glucose transporters and Plg, thereby controlling the levels of blood glucose and insulin to maintain glucose homeostasis.

Animals ; Chlorogenic Acid ; pharmacology ; Diet, High-Fat ; adverse effects ; Glucagon-Like Peptide 1 ; metabolism ; Glucose ; metabolism ; Glucose Tolerance Test ; Glucose Transporter Type 2 ; metabolism ; Homeostasis ; Insulin ; blood ; Intestines ; drug effects ; metabolism ; Male ; Proglucagon ; metabolism ; Random Allocation ; Rats, Sprague-Dawley ; Sodium-Glucose Transporter 1 ; metabolism ; Weight Gain ; drug effects

OBJECTIVETo investigate the effect of liraglutide, an analogue of glucagon-like peptide-1, on the proliferation and migration of cardiac microvascular endothelial cells (CMECs) and explore the mechanism.

METHODSIn vitro cultured CMECs of SD rats were purified by differential adhesion method and identified immunocytochemically using CD31 antibody and factor VIII. MTT assay was performed to assess the proliferation of the first-generation cells exposed to different concentrations (0-1000 nm/L) of liraglutide. Western blotting was used to detect the activation of PI3K/Akt and MAPK/ERK signaling pathways. BrdU fluorescent labeling and scratch assay were performed to observe the proliferation and migration of CMECs following liraglutide treatment, and PI3K/Akt and MAPK/ERK pathway inhibitors LY294002 and PD98059, respectively, were used to further confirm the role of these signaling pathways in regulating the proliferation and migration of CMECs.

RESULTSImmunocytochemical staining demonstrated a proportion of double positive cells exceeding 95%. The cells exhibited a logarithmic growth 48 h after plating. Liraglutide exposure concentration-dependently promoted the proliferation of CMECs with the optimal concentration of 100 nmol/L (P<0.05). Liraglutide exposure of the cells for 24 h significantly increased the levels of intracellular phosphorylated Akt and ERK (P<0.05), but pretreatment of the cells with Akt and ERK signaling pathway inhibitors 1 h before liraglutide obviously reversed such effect (P<0.05). BrdU and scratch assay showed that 100 nmol/L liraglutide significantly promoted the proliferation and migration of CMECs (P<0.05), but such effects were obviously suppressed by Akt and ERK inhibitors (P<0.05).

CONCLUSIONLiraglutide promotes the proliferation and migration of CMECs in vitro via PI3K/Akt and MAPK/ERK signaling pathways.

Animals ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Chromones ; Endothelial Cells ; cytology ; drug effects ; Flavonoids ; Glucagon-Like Peptide 1 ; analogs & derivatives ; pharmacology ; Liraglutide ; MAP Kinase Signaling System ; Morpholines ; Myocardium ; cytology ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; Rats ; Rats, Sprague-Dawley

Non-alcoholic fatty liver disease (NAFLD) is a common liver disease and it represents the hepatic manifestation of metabolic syndrome, which includes type 2 diabetes mellitus (T2DM), dyslipidemia, central obesity and hypertension. Glucagon-like peptide-1 (GLP-1) analogues and dipeptidyl peptidase-4 (DPP-4) inhibitors were widely used to treat T2DM. These agents improve glycemic control, promote weight loss and improve lipid metabolism. Recent studies have demonstrated that the GLP-1 receptor (GLP-1R) is present and functional in human and rat hepatocytes. In this review, we present data from animal researches and human clinical studies that showed GLP-1 analogues and DPP-4 inhibitors can decrease hepatic triglyceride (TG) content and improve hepatic steatosis, although some effects could be a result of improvements in metabolic parameters. Multiple hepatocyte signal transduction pathways and mRNA from key enzymes in fatty acid metabolism appear to be activated by GLP-1 and its analogues. Thus, the data support the need for more rigorous prospective clinical trials to further investigate the potential of incretin therapies to treat patients with NAFLD.
Animals ; Clinical Trials as Topic ; Dipeptidyl-Peptidase IV Inhibitors ; pharmacology ; therapeutic use ; Glucagon-Like Peptide 1 ; agonists ; Humans ; Hypoglycemic Agents ; pharmacology ; therapeutic use ; Lipid Metabolism ; drug effects ; Non-alcoholic Fatty Liver Disease ; drug therapy ; metabolism ; Triglycerides ; metabolism