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Glucagon-like peptide-1 (GLP-1) is expressed in retinal neurons, but its role in the retina is largely unknown. Here, we demonstrated that GLP-1 or the GLP-1 receptor (GLP-1R; a G protein-coupled receptor) agonist exendin-4 suppressed γ-aminobutyric acid receptor (GABAR)-mediated currents through GLP-1Rs in isolated rat retinal ganglion cells (GCs). Pre-incubation with the stimulatory G protein (Gs) inhibitor NF 449 abolished the exendin-4 effect. The exendin-4-induced suppression was mimicked by perfusion with 8-Br-cAMP (a cAMP analog), but was eliminated by the protein kinase A (PKA) inhibitor Rp-cAMP/KT-5720. The exendin-4 effect was accompanied by an increase in [Ca2+]i of GCs through the IP3-sensitive pathway and was blocked in Ca2+-free solution. Furthermore, when the activity of calmodulin (CaM) and CaM-dependent protein kinase II (CaMKII) was inhibited, the exendin-4 effect was eliminated. Consistent with this, exendin-4 suppressed GABAR-mediated light-evoked inhibitory postsynaptic currents in GCs in rat retinal slices. These results suggest that exendin-4-induced suppression may be mediated by a distinct Gs/cAMP-PKA/IP3/Ca2+/CaM/CaMKII signaling pathway, following the activation of GLP-1Rs.
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Objective To explore the effect of Exendin-4 on dementia after traumatic brain injury (TBI) in rats and its related mechanism.Methods Thirty SD rats were randomly divided into sham group (n=10),TBI group(n=10) and Exendin-4 group(n=10).Cortical impact injury was used to construct the TBI model.Morris water maze test was used to test the memory function of rats one month after TBI.The beta-amyloid protein (Aβ1-42) and nuclear factor erythroid-2-related factor 2 (Nrf2) were detected by Western blot.Results One month after TBI compared with the sham group,the escape latency (EL) ((35.31 ± 13.23)s vs (8.79±9.71)s) was prolonged and the target quadrant stay time ((17.78±4.68)s vs (26.35± 5.83)s) was shortened,the number of crossing platforms ((1.40±1.75) vs (3.50±1.45)) decreased,the relative content of Aβ1-42 in hippocampus ((1.0140±0.0328) vs (0.4355±0.0152)) increased the relative content of tau protein ((0.8039±0.0251) vs (0.5170±0.0185)) increased,and Nrf2 expression levels ((0.3851±0.0188) vs (0.4901±± 0.0140)) decreased significantly,and the differences were statistically significant (t=5.110,3.625,4.068,16.010,9.208,4.474,all P＜0.01);Compared with TBI group,EL ((23.74±10.95) vs (35.31±13.23)) shortened,target quadrant dwell time ((24.28±5.37) vs (17.78±± 4.68)) shortened,the number of crossing platforms ((3.30±1.88) vs (1.40±1.75)) decreased,and the relative content of Aβ1-42 in hippocampus ((0.8370±0.0188) vs (1.0140±0.0328)) significantly decreased,the relative content of tau protein ((0.6693±0.0166) vs (0.8039±0.0251)) significantly decreased,and the expression level of Nrf2 ((0.4738 ± 0.0166) vs (0.3851 ± 0.0188)) significantly increased,and the differences were statistically significance (t=2.052,2.866,5.196,4.693,3.480,3.538,all P＜0.01).Conclusion Exendin-4 can significantly improve the learning and memory function of TBI rats,increase the expression of Nrf2,decrease the content of Aβ1-42 and tau in hippocampus,and improve the prognosis of neurological function of TBI rats.
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OBJECTIVE@#To investigate the effects of exendin-4 on hepatic lipid metabolism, fibrosis and oxidative stress in mice with streptozotocin (STZ)-induced diabetes and explore the underlying mechanisms.@*METHODS@#C57BL/6J mice were fed with high-fat diet (HFD) for 4 weeks and received intraperitoneal injections of 120 mg/kg STZ to induce diabetes. After successful modeling, the mice were randomized into diabetic control group and exendin-4 treatment group (DM+E4), and in the latter group, the mice were given a daily dose of 1 nmol/kg of exendin-4 for 8 weeks. The changes in the body weight (BW) and random blood glucose (RBG) in the mice were recorded. The mRNA expressions of the genes related with liver lipid metabolism, fibrosis and oxidative stress were analyzed using RT-PCR, and the structural changes of the liver tissues were observed with HE, Sirius red and oil red O staining; the expressions of TGF-β1, Nrf2 and HO-1 proteins in the liver tissues were detected using Western blotting.@*RESULTS@#The diabetic mice showed significantly higher RBG levels and BW with obvious lipid deposition, fibrosis and oxidative stress in the liver as compared with the normal control mice ( < 0.001). Exendin-4 treatment of the diabetic mice did not significantly lessened liver lipid deposition but obviously reduced the levels of RBG and TG ( < 0.05), lowered the expression levels of liver fibrosis-related genes TGF-β, -SMA and Col-Ⅰ ( < 0.05), increased the expression levels of the antioxidant genes Nrf2, HO-1 and GPX4 ( < 0.01), and enhanced the protein expressions of Nrf2 and HO-1 in the liver tissues ( < 0.01).@*CONCLUSIONS@#Exendin-4 improves liver fibrosis and oxidative stress in diabetic mice by activating Nrf2/HO-1 pathway without significantly reducing liver lipid deposition.
Subject(s)
Animals , Mice , Diabetes Mellitus, Experimental , Exenatide , Liver , Liver Cirrhosis , Mice, Inbred C57BL , NF-E2-Related Factor 2 , Oxidative Stress , StreptozocinABSTRACT
Objective:To investigate the effects of Exendin-4 (Ex-4) on the expressions of lipid metabolism related genes in the human liver cancer HepG2 cells with insulin resistance (IR),and to elucidate the effect of Ex-4in improvement of IR.Methods:The HepG2 cells in logarithmic growth phase were induced into IR model with high concentration of insulin,then divided into control group (HepG2 cells),IR group (HepG2 cells were treated with insulin,HepG2-IR cells),and Ex-4 group (HepG2-IR cells were treated with Ex-4).Glucose oxidase (GOD-POD)kit was used to detect the consumption of glucose.The cell morphology and intracellular lipid drip formation were observed by Oil red O staining.The triglyceride (TG) level in cells was detected by kit;qRT-PCR was used to detect the mRNA expression levels of acetyl-CoA carboxylase (ACC),fatty acid synthase (FAS),sterol regulatory element-binding protein-1c (SREBP-1c) and apolipoprotein B100 (apoB100).Results:Compared with control group (HepG2 cells),the glucose consumption in the HepG2-IR cells in IR group was significantly decreased (P＜0.01).Compared with IR group,the glucose consumption in the HepG2-IR cells in Ex-4 group was increased (P＜0.05).The Oil O red staining results showed that compared with control group,the fat percentage in the HepG2-IR cells in IR group was increased (P＜0.05);compared with IR group,the fat percentage in Ex-4 group was decreased (P＜0.05).Compared with control group,the level of TG in the cells in IR group was significantly increased (P＜0.01);compared with IR group,the level of TG in the cells in Ex-4 group was significantly decreased (P＜0.05).The qT-PCR results showed that compared with control group,the expression levels of ACC FAS and SREBP-1cmRNA in the cells in IR group were increased (P＜0.01),and the expression level of apoB100 mRNA was decreased (P＜0.05);compared with IR group,the expression levels of ACC,FAS and SREBP-1c mRNA in the cells in Ex-4 group were decreased (P＜0.05),and the expression level of apoB100 mRNA was increased (P＜0.01).Conclusion:Ex-4 can regulate the expressions of lipid metabolism related genes in the HepG2 cells and improve IR.
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Objective To explore whether exendin-4 inhibits AR42J cells and its mechanism.Methods AR42J cells were treated with exendin-4 under multiple concentrations(1,5,10 pmol/L) at 24,48,72,96,120 h to assess its cell viability by MTT assay and got the IC-50 and time points.Then checking whether exendin-4 could induce the AR42Jcells apoptosis by setting normal control (NC) group,exendin-4 (Ex-4) group and Ex-4+ z-VAD-fmk (apoptosis inhibitor) group,and exploring whether 3-MA which is autophagy inhibitor could inhibit the AR42J cells apoptosis induced by exendin-4 by setting NC group,Ex-4 group and Ex-4+3-MA group.Cell viability was analyzed by MTT and the cells apoptosis was detected by flow cytometry and the protein levels of caspase-3,LC3 and p62 were studied by Western blot.Results Concentration of 10 pmol/L exendin-4 and and time point 72 h were selected for the further study.z-VAD-fmk pretreatment can significantly inhibit the cell viability of exendin-4 by (81.2±3.3)％ vs.(49.4±3.0)％ (P＜0.05).Flow cytometry showed that exendin-4 could induce the AR42J cells apoptosis by (28.2± 1.4)％ vs.(3.6±0.8)％,and increased the caspase-3 level by Western blot,which both can be reversed by (79.1 ±2.3) ％ vs.(49.8±2.5)％ (P＜0.05) when the cells were treated for 72 h,as was apoptosis ratio by (14.5±2.1)％ vs.(29.2±3.2)％.Western blot showed that exendin-4 can upregulate protein levels of LC3B-Ⅱ,p62 和 caspase-3 and 3-MA,and pretreatment can inhibit the upregulation of LC3B-Ⅱ and caspase-3 but further increased the upregulation of p62 induced by exendin-4.Conclusions Exendin-4 can induce AR42J cells apoptosis and 3-MA pretreating can inhibit exendin-4 cytotoxicity through downregulating autophagy.So auto phagy inhibitor 3-MA could potentially extenuate the cytotoxicity of exendin-4 in pancreatic acinar cells.
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Objective: To investigate the effects of Exendin-4 (Ex-4) on the expressions of lipid metabolism related genes in the human liver cancer HepG2 cells with insulin resistance (IR), and to elucidate the effect of Ex-4 in improvement of IR. Methods: The HepG2 cells in logarithmic growth phase were induced into IR model with high concentration of insulin, then divided into control group (HepG2 cells), IR group (HepG2 cells were treated with insulin, HepG2-IR cells), and Ex-4 group (HepG2-IR cells were treated with Ex-4). Glucose oxidase (GOD-POD) kit was used to detect the consumption of glucose. The cell morphology and intracellular lipid drip formation were observed by Oil red O staining. The triglyceride (TG) level in cells was detected by kit; qRT-PCR was used to detect the mRNA expression levels of acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), sterol regulatory element-binding protein-1c (SREBP-lc) and apolipoprotein B100 (apoBlOO). Results: Compared with control group (HepG2 cells), the glucose consumption in the HepG2-IR cells in IR group was significantly decreased (P<0. 01). Compared with IR group, the glucose consumption in the HepG2-IR cells in Ex-4 group was increased (P<0. 05). The Oil O red staining results showed that compared with control group, the fat percentage in the HepG2-IR cells in IR group was increased (P<0. 05); compared with IR group, the fat percentage in Ex-4 group was decreased (P< 0.05). Compared with control group, the level of TG in the cells in IR group was significantly increased (P< 0. 01); compared with IR group, the level of TG in the cells in Ex-4 group was significantly decreased (P<0. 05). The qT-PCR results showed that compared with control group, the expression levels of ACC FAS and SREBP-lc mRNA in the cells in IR group were increased (P<0. 01), and the expression level of apoBlOO mRNA was decreased (P<0. 05); compared with IR group, the expression levels of ACC, FAS and SREBP-lc mRNA in the cells in Ex-4 group were decreased (P<0. 05), and the expression level of apoBlOO mRNA was increased (P< 0.01). Conclusion: Ex-4 can regulate the expressions of lipid metabolism related genes in the HepG2 cells and improve IR.
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AIM: To explore the effects of exendin-4 (EX-4) on endoplasmic reticulum stress (ERS)-mediated insulin resistance in the 3T3-L1 adipocytes.METHODS: In vitro 3T3-L1 pre-adipocytes were differentiated into adipocytes, and the cells were treated with tunicamycin (TM), tauroursodeoxycholic acid (TUDCA) or EX-4, respectively.The cell viability was measured by MTT assay.The glucose consumption was determined by glucose oxidase assay to evaluate insulin sensitivity of the 3T3-L1 adipocytes with different interventions.The protein levels of p-Akt, Akt and endoplasmic reticulum stress markers, including inositol requiring enzyme-1 (IRE1), p-IRE1, JNK, p-JNK, protein kinase R-like endoplasmic reticulum kinase (PERK), p-PERK, eukaryotic initation factor 2 alpha (eIF2a), p-eIF2a, activating transcription factor-6(ATF-6) were detected by Western blot.RESULTS: The insulin-stimulated glucose consumption and the protein level of p-Akt were inhibited by TM at 5 mg/L for 5 h (P<0.05), while they were increased when the cells were treated with TUDCA at 1 mmol/L or EX-4 at 100 nmol/L for 24 h (P<0.05).The effects above induced by TM (5 mg/L for 5 h) were also blunted by pretreating with TUDCA at 1 mmol/L or EX-4 at 100 nmol/L for 24 h (P<0.05).The protein levels of ERS markers such as p-IRE1, p-JNK, p-PERK, p-eIF2a and ATF-6 were significantly increased by treating with TM at 5 mg/L for 5 h, whereas 24 h pre-treatment with TUDCA or Ex-4 alleviated the ERS of the 3T3-L1 adipocytes induced by TM.The expression of total IRE1, JNK, PERK and eIF2a was not changed in different groups.CONCLUSION: Exendin-4 improves endoplasmic reticulum stress mediated insulin resistance in 3T3-L1 adipocytes.
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Objective To explore the effects and related mechanism of Exendin-4 on secretion of extracellular matrix in high glucose-induced glomerular mesangial cells(GMCs). Methods GMCs were incubated in medium with glucose or Exendin-4 for the four groups: normal glucose group(NG group): cells were treated in medium with 5.6 mmol/L glucose; NG with Exendin-4 treatment group(NGE group): cells were treated with 5.6 mmol/L glucose and Exendin-4; high glucose group(HG group): cells were cultured with 30 mmol/L glucose; HG with Exendin-4 treatment group(HGE group): cells were treated with 30 mmol/L glucose and Exendin-4 at concentration of 3, 5, 10, 15, 30 nmol/L separately, which were cultured for 12, 24, 48 hours. GMCs treated with Exendin-4 were determined by assessing proliferation using a CCK8 method. The levels of fibronectin(FN), collagen type Ⅳ(Col-Ⅳ)in the cell supernatant were measured using enzyme-linked immunosorbent assay(ELISA). The gene levels of Col-Ⅳ, FN, and expression of inflammatory mediators including monocyte chemotactic protein 1(MCP-1), tumor necrosis factor-α(TNF-α), intercellular cell adhesion molecule-1(ICAM-1), transforming growth factor-β1(TGF-β1)were evaluated using reverse transcription PCR(RT-PCR); The expression of nuclear transcription factor-κB(NF-κB), glucagon-like peptide-l receptor(GLP-1R), and phosphorylation levels of mitogen-activated protein kinases(MAPKs)were evaluated by Western blot. Results(1)After treatment with 10 nmol/L Exendin-4 for 24 hour, the proliferation rate of GMCs was significantly decreased compared with 3 nmol/L, 5 nmol/L Exendin-4 treatment(P0.05). (2)The gene expression of FN, Col-Ⅳ and the inflammatory mediators, MCP-1, TNF-α, ICAM-1, TGF-β1 in HG group were significantly increased compared with the NG group,(all P<0.05). After treatment with Exendin-4, the levels of FN, Col-Ⅳ and the gene expression of TNF-α, MCP-1, ICAM-1, TGF-β1 were decreased(all P<0.05). (3)Compared with NG group, the expression of NF-κB and the phosphorylation of extracellular regulated protein kinases(p-Erk1/2), Jun N-terminal kinase(p-JNK)and, p38MAPK(p-p38MAPK)in the group of HG group were increased significantly, accompanied by the decrease of GLP-1R protein level(all P<0.05). Importantly, Exendin-4 treatment significantly reduced protein expression of p-Erk1/2, p-JNK, and NF-κB(all P<0.05), and the level of GLP-1R protein increased(P<0.05). Furthermore, specific Erk1/2, JNK or NF-κB inhibitors markedly blocked Exendin-4-mediated decrease in the levels of FN, Col-Ⅳ. Conclusion Exendin-4 treatment inhibits the secretion of extracellular matrix potentially through Erk1/2, JNK/NF-κB signaling in higher glucose induced GMCs.
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@#The assay method of GLP-1 receptor binding affinity for a long-acting hypoglycemic peptide—PEgylated Exendin-4 analogue(PE)was optimized and established based on the luciferase reporter gene approach. CHO-GLP-1R-CRE-Luc+ cells were previously constructed in our lab followed by the verification of methodology. This assay method showed good specificity and robustness as well as high accuracy and precision when PE was incubated with the cell for 4 h, the luminescent substrate reacted with cell lysates for 15 min and the concentration for PE ranged 5. 7×10-3-1. 5×103 nmol/L, on which condition this developed method is in accordance with General Principles of Analytical Method Validation Techniques for Biological Products Quality Control. This study also lays the foundations for rapid evaluation and screening of GLP-1 receptor agonist drugs.
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Objective To study the effect of Exendin-4 on oxidative stress and neural apoptosis following spinal cord injury (SCI). Methods Adult male SD rats, with weight between 200-250 g, were randomly divided into three groups (12 in each group):Sham group, SCI group and Exendin-4 group (Ex-4 group). Rats in Sham group achieved spinal cord exposure. SCI group and Ex-4 group were induced according to Allen′s test (using a weight-drop device). Rats in Ex-4 group were ad?ministrated with Exendin-4 (10 μg/rat) through intraperitoneal injection immediately after establishment of SCI models. Rats in Sham group and SCI group were given the same volume of normal saline solution instead. Level of malondialdehyde (MDA) and the activity of catalase (CAT) were assessed in spinal cord tissues 24 hour after drug administrations. Neural apoptosis was detected by TUNEL staining and the expression levels of caspase-9 and AIF were determined using Western blot. Results Compared with Sham group, the levels of MDA, caspase-9 and AIF as well as neuronal apoptosis rate in?creased obviously, while activity of CAT decreased markedly in SCI group(P<0.01). Compared with SCI group, the levels of MDA, caspase-9 and AIF as well as the neuronal apoptosis rate decreased obviously, while activity of CAT increased re?markably in Exendin-4 group(P < 0.01). Conclusion Exendin-4 restrain neural apoptosis following spinal cord injury through relieving oxidative damage.
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BACKGROUND: Selenoprotein P (SEPP1) and fetuin-A, both circulating liver-derived glycoproteins, are novel biomarkers for insulin resistance and nonalcoholic fatty liver disease. However, the effect of exendin-4 (Ex-4), a glucagon-like peptide-1 receptor agonist, on the expression of hepatokines, SEPP1, and fetuin-A, is unknown. METHODS: The human hepatoma cell line HepG2 was treated with palmitic acid (PA; 0.4 mM) and tunicamycin (tuni; 2ug/ml) with or without exendin-4 (100 nM) for 24 hours. The change in expression of PA-induced SEPP1, fetuin-A, and endoplasmic reticulum (ER) stress markers by exendin-4 treatment were evaluated using quantitative real-time reverse transcription polymerase chain reaction and Western blotting. Transfection of cells with AMP-activated protein kinase (AMPK) small interfering RNA (siRNA) was performed to establish the effect of exendin-4-mediated AMPK in the regulation of SEPP1 and fetuin-A expression. RESULTS: Exendin-4 reduced the expression of SEPP1, fetuin-A, and ER stress markers including PKR-like ER kinase, inositol-requiring kinase 1alpha, activating transcription factor 6, and C/EBP homologous protein in HepG2 cells. Exendin-4 also reduced the expression of SEPP1 and fetuin-A in cells treated with tunicamycin, an ER stress inducer. In cells treated with the AMPK activator 5-aminoidazole-4-carboxamide ribonucleotide (AICAR), the expression of hepatic SEPP1 and fetuin-A were negatively related by AMPK, which is the target of exendin-4. In addition, exendin-4 treatment did not decrease SEPP1 and fetuin-A expression in cells transfected with AMPK siRNA. CONCLUSION: These data suggest that exendin-4 can attenuate the expression of hepatic SEPP1 and fetuin-A via improvement of PA-induced ER stress by AMPK.
Subject(s)
Humans , Activating Transcription Factor 6 , alpha-2-HS-Glycoprotein , AMP-Activated Protein Kinases , Blotting, Western , Carcinoma, Hepatocellular , Cell Line , Endoplasmic Reticulum , Endoplasmic Reticulum Stress , Fatty Liver , Glucagon-Like Peptide 1 , Glycoproteins , Hep G2 Cells , Insulin Resistance , Palmitic Acid , Phosphotransferases , Polymerase Chain Reaction , Reverse Transcription , RNA, Small Interfering , Selenoprotein P , Transfection , Tunicamycin , Biomarkers , Glucagon-Like Peptide-1 ReceptorABSTRACT
OBJECTIVE: To prepare poly(lactic-co-glycolic acid) (PLGA) microspheres of Exendin-4 and evaluate its characteristics. METHODS: Exendin-4 microspheres were prepared by different preparation methods (W/O/W, S/O/O and S/O/W method) and characterized for encapsulation efficiency by high-performance liquid chromatography (HPLC), particle size by laser particle size analyzer, surface morphology by scanning electron microscopy (SEM), and Exendin-4 stability by circular dichroism (CD). Release rate in vitro was determined, and the microspheres were administered subcutaneously to type 2 diabetic rats to determine its pharmacokinetics and pharmacodynamics. RESULTS: Exendin-4 was successfully encapsulated into PLGA microspheres by W/O/W method. The average size was 25-30 μm. Exendin-4 encapsulation efficiency was 82.2%. CD confirmed that the structural integrity of the peptide was intact. The burst release in 24 h was 15.2% and the cumulated release in 28 d was up to 90% in vitro. Animal study demonstrated the drug release from microspheres in vivo and in vitro had a good correlation(r=0.9623) and the blood glucose level was controlled for 4 weeks by Exendin-4 microspheres. CONCLUSION: The PLGA microspheres can be used for the controlled delivery of Exendin-4.
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Exendin-4 (Ex-4), a glucagon-like peptide-1 receptor (GLP-1R) agonist, has been known to reverse hepatic steatosis in ob/ob mice. Although many studies have evaluated molecular targets of Ex-4, its mechanism of action on hepatic steatosis and fibrosis has not fully been determined. In the liver, glucose transporter 4 (GLUT4) is mainly expressed in hepatocytes, endothelial cells and hepatic stellate cells (HSCs). In the present study, the effects of Ex-4 on GLUT4 expression were determined in the liver of ob/ob mice. Ob/ob mice were treated with Ex-4 for 10 weeks. Serum metabolic parameters, hepatic triglyceride levels, and liver tissues were evaluated for hepatic steatosis. The weights of the whole body and liver in ob/ob mice were reduced by long-term Ex-4 treatment. Serum metabolic parameters, hepatic steatosis, and hepatic fibrosis in ob/ob mice were reduced by Ex-4. Particularly, Ex-4 improved hepatic steatosis by enhancing GLUT4 via GLP-1R activation in ob/ob mice. Ex-4 treatment also inhibited hepatic fibrosis by decreasing expression of connective tissue growth factor in HSCs of ob/ob mice. Our data suggest that GLP-1 agonists exert a protective effect on hepatic steatosis and fibrosis in obesity and type 2 diabetes.
Subject(s)
Animals , Mice , Connective Tissue Growth Factor , Endothelial Cells , Fatty Liver , Fibrosis , Glucagon-Like Peptide 1 , Glucagon-Like Peptide-1 Receptor , Glucose Transport Proteins, Facilitative , Hepatic Stellate Cells , Hepatocytes , Liver , Obesity , Triglycerides , Weights and MeasuresABSTRACT
The aim of this study was to evaluate Exendin-4 (EX-4) effects on islet volume and number in the mouse pancreas. Thirty-two healthy adult male NMRI mice were randomly divided into control and experimental groups. EX-4 was injected intraperitoneally (i. p.) at doses of 0.25 (E1 group), 0.5 (E2 group), and 1 µg/kg (E3 group), twice a day for 7 consecutive days. One day after the final injection, the mice were sacrificed, and the pancreas from each animal dissected out, weighed, and fixed in 10% formalin for measurement of pancreas and islet volume, and determination of islet number by stereological assessments. There was a significant increase in the weight of pancreases in the E3 group. Islet and pancreas volumes in E1 and E2 groups were unchanged compared to the control group. The E3 group showed a significant increase in islet and pancreas volume (P < 0.05). There were no significant changes in the total number of islets in all three experimental groups. The results revealed that EX-4 increased pancreas and islet volume in non-diabetic mice. The increased total islet mass is probably caused by islet hypertrophy without the formation of additional islets.
O objetivo deste estudo foi avaliar os efeitos do Exendin-4 (EX-4) sobre o volume e número de ilhotas no pâncreas. Trinta e dois camundongos NMRI machos saudáveis e adultos foram divididos ao acaso em grupos controle e grupos experimentais. EX-4 foi injetado intraperitonealmente (i. p.) nas doses de 0,25 (grupo E1), 0,5 (grupo E2) e 1 (grupo E3), duas vezes por dia durante 7 dias consecutivos. Um dia após a injeção final, os camundongos foram sacrificados e o pâncreas de cada animal foi dissecado, pesado e fixado em solução de formaldeído 10% para avaliação do volume do pâncreas e ilhotas e do número de ilhotas por métodos estereológicos. Observou-se aumento significativo no peso de pâncreas no grupo E3. O volume do pâncreas assim como das ilhotas não apresentou alterações nos grupos E1 e E2, quando comparados ao grupo controle No grupo E3 houve aumento significativo no volume do pâncreas e das ilhotas (P<0,05). Não se observaram alterações significativas no número de ilhotas nos três grupos experimentais. Os resultados revelaram que o EX-4 provoca aumento no volume do pâncreas, bem como no volume das ilhotas em camundongos não-diabéticos. O aumento no volume total de ilhotas deve-se, provavelmente, a hipertrofia das ilhotas sem a formação de ilhotas adicionais.
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Rats , Rats/classification , Islets of Langerhans/physiology , Pancreas , Glucagon-Like Peptide-1 Receptor , Hypertrophy/classificationABSTRACT
The incidence of diabetes mellitus is increasing among companion animals. This disease has similar characteristics in both humans and animals. Diabetes is frequently identified as an independent risk factor for infections associated with increased mortality. In the present study, homozygous diabetic (db/db) mice were infected with Listeria (L.) monocytogenes and then treated with the anti-diabetic drug exendin-4, a glucagon-like peptide 1 analogue. In aged db/db mice, decreased CD11b+ macrophage populations with higher lipid content and lower phagocytic activity were observed. Exendin-4 lowered high lipid levels and enhanced phagocytosis in macrophages from db/db mice infected with L. monocytogenes. Exendin-4 also ameliorated obesity and hyperglycemia, and improved ex vivo bacteria clearance by macrophages in the animals. Liver histology examined during L. monocytogenes infection indicated that abscess formation was much milder in exendin-4-treated db/db mice than in the control animals. Moreover, mechanistic studies demonstrated that expression of ATP binding cassette transporter 1, a sterol transporter, was higher in macrophages isolated from the exendin-4-treated db/db mice. Overall, our results suggest that exendin-4 decreases the risk of infection in diabetic animals by modifying the interaction between intracellular lipids and phagocytic macrophages.
Subject(s)
Animals , Female , Mice , ATP-Binding Cassette Transporters/metabolism , Age Factors , Blood Chemical Analysis , Cholesterol/metabolism , Diabetes Mellitus, Type 2/drug therapy , Dyslipidemias/drug therapy , Hyperglycemia/drug therapy , Hypoglycemic Agents/therapeutic use , Injections, Intraperitoneal , Lipid Metabolism/drug effects , Listeria monocytogenes/drug effects , Listeriosis/drug therapy , Macrophages/drug effects , Obesity/drug therapy , Peptides/therapeutic use , Phagocytosis/drug effects , Venoms/therapeutic useABSTRACT
The present study has been undertaken to investigate the effect of exendin-4 (a glucagon-like peptide-1 agonist) in diabetes mellitus (DM) and hyperhomocysteinemia (HHcy)-induced vascular endothelial dysfunction (VED). Streptozotocin (55 mg kg−1, iv, once) and methionine (1.7% w/w, po, 4 weeks) were administered to rats to produce DM (serum glucose >200 mg dl−1) and HHcy (serum homocysteine >10 μM) respectively. VED was assessed using isolated aortic ring preparation, microscopy of thoracic aorta, and serum nitrite/nitrate concentration. Serum TBARS concentration was estimated to assess oxidative stress. Atorvastatin has been employed as standard agent. Exendin-4 (1 μg kg−1, ip) and atorvastatin (30 mg kg−1, po) treatments significantly attenuated increase in serum glucose and homocysteine but their concentrations remained markedly higher than sham control value. Exendin-4 and atorvastatin treatments markedly prevented DM and HHcy-induced (i) attenuation of acetylcholine-induced endothelium-dependent relaxation, (ii) impairment of vascular endothelial lining, (iii) decrease in serum nitrite/nitrate concentration, and (iv) increase in serum TBARS. However, this ameliorative effect of exendin-4 has been prevented by L-NAME (25 mg kg-1, ip), an inhibitor of NOS. It may be concluded that exendin-4 may activate eNOS due to activation of GLP-1 and consequently reduce oxidative stress to improve vascular endothelial dysfunction.
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Oxidative stress induced by chronic hyperglycemia in type 2 diabetes plays a crucial role in progressive loss of beta-cell mass through beta-cell apoptosis. Glucagon like peptide-1 (GLP-1) has effects on preservation of beta-cell mass and its insulin secretory function. GLP-1 possibly increases islet cell mass through stimulated proliferation from beta-cell and differentiation to beta-cell from progenitor cells. Also, it probably has an antiapoptotic effect on beta-cell, but detailed mechanisms are not proven. Therefore, we examined the protective mechanism of GLP-1 in beta-cell after induction of oxidative stress. The cell apoptosis decreased to ~50% when cells were treated with 100 microM H2O2 for up to 2 hr. After pretreatment of Ex-4, GLP-1 receptor agonist, flow cytometric analysis shows 41.7% reduction of beta-cell apoptosis. This data suggested that pretreatment of Ex-4 protect from oxidative stress-induced apoptosis. Also, Ex-4 treatment decreased GSK3beta activation, JNK phosphorylation and caspase-9, -3 activation and recovered the expression of insulin2 mRNA in beta-cell lines and secretion of insulin in human islet. These results suggest that Ex-4 may protect beta-cell apoptosis by blocking the JNK and GSK3beta mediated apoptotic pathway.
Subject(s)
Animals , Cricetinae , Humans , Apoptosis , Caspase 3/metabolism , Caspase 9/metabolism , Cells, Cultured , Flow Cytometry , Glucagon-Like Peptide 1/pharmacology , Glycogen Synthase Kinase 3/metabolism , Hydrogen Peroxide/toxicity , Insulin/genetics , Insulin-Secreting Cells/drug effects , JNK Mitogen-Activated Protein Kinases/metabolism , Oxidative Stress , Peptides/pharmacology , Phosphorylation , Receptors, Glucagon/agonists , Signal Transduction , Venoms/pharmacologyABSTRACT
AIM To investigate the tissue distribution of exendin-4 after administration in healthy rats. METHODS Exendin-4 was radioiodinated by the Iodo-GenTMmethod. Tissue distribution of [125I]exendin-4 was investigated after sc administration of [125I]exendin-4 at 3 μg·kg-1 in rats. Both total radioactivity and trichloroacetic acid (TCA) precipitated radioactivity were used to calculate the levels of [125I]exendin-4 in rats plasma and tissue samples after sc administration. RESULTS The tissue distribution of [125I]exendin-4 after sc injection showed substantial disposition in kidneys, lungs, bladder and pancreas. The rank order of normalized tissue distribution was kidneys＞lungs＞bladder＞pancreas＞intestine＞plasma＞adrenals>jejunum＞lymph＞liver＞spleen＞heart＞marrow＞thymus＞testicles＞brain＞muscle＞adipose. CONCLUSION [125I]Exendin-4 underwent a rapid and wide distribution in the tissues throughout the whole body within the time course examined. TCA precipitated radioactivity in kidneys was the highest, however, only trace amounts of [125I]exendin-4 was detected in the brain.
ABSTRACT
Objective To investigate the effects of exendin-4 (exenatide) on insulin sensitivity and adipocytokine in high-fat-fed rats. Methods Rats were divided randomly into normal-chow group (NC), high-fat group (HF) and high-fat+exendin treated group (HE). HE rats were given exenatide (2 μg/kg) twice daily for 6 wk. The insulin sensitivity was evaluated by intravenous insulin tolerance test (IVITT). Insulin-stimulated changes in insulin signal transduction, visfatin and adiponectin mRNA expressions as well as their plasma levels were also observed in these rats. Results Plasma free fatty acids (FFA), triglyceride (TG), total cholesterol (TC) levels were significantly reduced after exenatide treatment (in HE rats all P<0.01). And IVITT parameters were also improved in these rats. Insulin-stimulated IRS-1 tyrosine phosphorylation was slightly increased in exenatide-treated rats as compared with HF rats (P<0.05). In addition,plasma visfatin level was significantly reduced in HF and HE groups as compared with controls (P<0.05 and P<0.01). The adiponectin mRNA expression in adipose tissues and circulating adiponectin level were significantly elevated in exenatide-treated rats as compared with untreated rats and controls (P<0.01). Conclusions Chronic exenatide treatment improves insulin resistance in high-fat-fed rats, and the changes of IRS-1 tyrosine phosphorylation and adiponectin may be related to the role of exenatide in elevating insulin sensitivity
ABSTRACT
BACKGROUND: The characteristic feature of pancreatic beta cells is highly developed endoplasmic reticulum (ER) due to a heavy engagement in insulin secretion. The ER serves several important function, including post-translational modification, folding, and assembly of newly synthesized secretory proteins, and its proper function is essential to cell survival. Various stress conditions can interfere with ER function. Pancreatic beta cells may be particularly vulnerable to ER stress that causes to impair insulin biosynthesis and beta cell survival through apoptosis. Glucagon like peptide-1 (GLP-1) is a new drug for treatment of type 2 diabetes and has effects on stimulation of insulin secretion and beta cell preservation. Also, it may have an antiapoptotic effect on beta cells, but detailed mechanisms are not proven. Therefore, we investigated the protective mechanism of GLP-1 in beta cells through ER stress response induced by 2-deoxy-D-glucose (2DG). METHODS: For induction of the ER stress, HIT-T15 cells (hamster beta cell line) were treated with 2DG (10 mM). Apoptosis was evaluated with MTT assay, hoechst 33342 staining and Annexin/PI flow cytometry. Expression of ER stress-related molecules was determined by real-time PCR or western blot. For blocking ER stress, we pretreated HIT-T15 cells with exendin-4 (Ex-4; GLP-1 receptor agonist) for 1 hour before stress induction. RESULTS: After induction with ER stress (2DG), beta cells were lost by apoptosis. We found that Ex-4 had a protective effect through ER stress related molecules (GRP78, GRP94, XBP-1, eIF2alpha, CHOP) modulation. Also, Ex-4 recovered the expression of insulin2 mRNA in beta cells. CONCLUSION: These results suggest that GLP-1 may protect beta cells apoptosis through ER stress modulation.