Abstract 13502: Glucagon-like Peptide-1 Directly Promotes Angiogenesis via PKA/AMPK-dependent Autophagy in Endothelial Cells

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Akio Monij ◽  
Yasuko K Bando ◽  
Morihiko Aoyama ◽  
Haruya Kawase ◽  
Toko Mitsui ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Capillary Density ◽  
Tube Formation ◽  
Catalytic Subunit ◽  
Related Gene ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
The Impact ◽  
In Vitro Angiogenesis

Introduction: We recently reported the impact of glucagon-like peptide-1 (GLP-1) on myocardial remodeling observed in type 2 diabetic mice (T2DM) via cyclic-AMP-dependent activation of autophagy in myocardium; however, it remains unclear whether GLP-1 may modulates capillary formation in heart. Hypothesis/AIM: We thus evaluated the impact of GLP-1 on angiogenesis and its link to endothelial autophagy. Methods: T2DM was treated with Ex4 (24 nmole/kg/day for 4 weeks). Cardiac capillary density was measured by immunohistology. Cultured HUVECs were used for in vitro experiments. Changes in activities of autophagy (LC3-turnover assay and protein levels of p62 and Beclin1), and angiogenesis (tube formation assay and Akt/AMPK/eNOS activity), were evaluated.[[Unable to Display Character: 
]] Role of PKA was assessed by CREB phosphorylation and RNA interference (siRNA for catalytic subunit of PKA). Effect of autophagy was assessed by use of pharmacological inhibitor 3MA and siRNA of autophagy-related gene (ATG) 5 , ATG7, and p62. Results: Immunohistochemical analyses revealed that T2DM exhibited reduced cardiac capillary density, which was reversed by Ex-4 with concomitant amelioration of systemic diabetic condition. Ex-4 treated heart exhibited increase in myocardial cyclic AMP concentration. We thus observed direct impact of Ex-4 and cyclic AMP elevation on HUVECs, in which GLP-1 receptor expression was confirmed by immunoblot and QPCR. In vitro angiogenesis assay revealed that Ex-4 and PKA enhancers (10 μM forskolin and 1 mM 8-bromo-cyclic AMP) facilitated angiogenesis and autophagy in HUVECs. The PKA/AMPK/eNOS phosphorylation levels of Ex-4-treated HUVECs were elevated. Of note, each Akt activity remains unchanged. PKA inhibitors (H89, RP-cAMP, siRNA) abrogated the increase in phosphorylation of AMPK/eNOS axis induced by Ex-4 in HUVECs. Tube formation assay revealed that Ex-4 and PKA enhancers augmented in vitro angiogenesis, which were abrogated by inhibition of autophagy and AMPK using pharmacological inhibitors (3MA and compound C) and siRNA for autophagy-related gene (ATG) 5 , ATG7, p62, and catalytic subunit of AMPK. [[Unable to Display Character: 
]] Conclusions: GLP-1 directly promoted angiogenesis via the PKA/AMPK-dependent autophagic activation.

Abstract 146: Glucagon-like Peptide-1 Receptor Activation Promotes Dual Benefits For Reversing Microvasculopathy And Mitochondrial Damage In Diabetic Heart.

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Akio Monji ◽  
Yasuko K Bando ◽  
Toko Mitsui ◽  
Morihiko Aoyama ◽  
Hiroya Kawase ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Receptor Activation ◽  
Left Ventricular ◽  
Mitochondrial Damage ◽  
Wall Thickening ◽  
Mouse Heart ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
The Impact

PURPOSE: Glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 (Ex4) is a remedy for type 2 diabetes mellitus (T2DM). Ex4 ameliorates cardiac dysfunction in preclinical and clinical settings. However, it remains unclear whether the impact of Ex4 on cardiac remodeling in diabetic cardiomyopathy (DMC), of which primary characteristics are microvasculopathy and mitochondrial damage. Methods and Results: Diet-induced T2DM (DIO) mice and age- and gender-matched lean control mice were allocated into EX4 (24 nmole/kg/day for 40 days; DIO-Ex4 and LEAN-Ex4) and vehicle groups (DIO-veh and LEAN-veh). We first confirmed the GLP-1R expression in every single chamber of mouse heart by immunoblotting and PCR. Ex4 treatment ameliorated both systemic and cardiac insulin resistance without affecting body weight in DIO. Cardiac capillary density of DIO-veh was reduced compared to those LEAN-veh, which were reversed by Ex4 treatment. Tube formation assay and immunoblot analysis using culture endothelial cells revealed that Ex4 directly enhanced in vitro angiogenesis in a PKA/eNOS-dependent fashion. Systolic and diastolic left-ventricular (LV) dysfunctions observed in DIO-veh were restored by Ex4 with decline in LV wall thickening. Myocardial fibrosis detected using sirius-red staining and tissue oxidative stress detected by a fluorescence indicator DHE were attenuated in DIO-Ex4. Of note, analyses using transmission electron microscopy and a fluorescence indicator for damaged mitochondria (mitotracker red) revealed that Ex4 treatment reversed cardiac mitochondrial remodeling and increased healthy mitochondria. Ex4 treatment modulated cardiac oxidative stress balance by upregulating antioxidative molecules (SOD, thioredoxin, glutathione peroxidase) and reduction of NOX4 level; whereas it had no influence on NOX2 level. Conclusions: Ex4 enhances cardiac angiogenesis via GLP-1R-mediated activation of PKA/eNOS axis and accelerates reverses remodeling of myocardial mitochondria, at least in part, via its facilitating effects on antioxidative defense.

Binding specificity and signal transduction of receptors for glucagon-like peptide-1(7–36)amide and gastric inhibitory polypeptide on RINm5F insulinoma cells

1993 ◽  
Vol 10 (3) ◽  
pp. 259-268 ◽  
Author(s):  
B Gallwitz ◽  
M Witt ◽  
U R Fölsch ◽  
W Creutzfeldt ◽  
W E Schmidt
Keyword(s):  
Cyclic Amp ◽  
Gastric Inhibitory Polypeptide ◽  
Dependent Manner ◽  
Binding Studies ◽  
Concentration Dependent Manner ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
Insulinoma Cells

ABSTRACT Glucagon-like peptide-1(7–36)amide (GLP-1(7–36) amide) and gastric inhibitory polypeptide (GIP), peptides of the glucagon family, stimulate insulin secretion in vitro and in vivo. They possess high N-terminal sequence homology. Binding studies with 125I-labelled GIP and 125I-labelled GLP-1(7– 36)amide were performed in RINm5F insulinoma cells to investigate receptor specificity and to compare both receptors directly. Both binding sites were highly ligand-specific: GIP did not bind to the GLP-1(7–36)amide receptor and vice versa. Both peptides increased intracellular cyclic AMP levels; GLP-1(7– 36)amide was 100-fold more potent in stimulating cyclic AMP production when compared with GIP. At ranges of 1–10 nmol GLP-1(7–36)amide/1 and 0·1–10 GIP/1, corresponding to submaximal binding concentrations, the hormones showed an additive effect on cyclic AMP production. The N-terminal portion of GIP was important for binding, as GIP(1–30) showed almost full binding and biological activity. GIP(17–42) bound in a concentration-dependent manner with approximately 500-fold lower potency than GIP. At concentrations of up to 10 μmol GIP(17–42)/1 no stimulation of cyclic AMP was observed.

scholarly journals Differences in signalling, trafficking and glucoregulatory properties of glucagon-like peptide-1 receptor agonists exendin-4 and lixisenatide

2019 ◽  
Author(s):  
Philip Pickford ◽  
Maria Lucey ◽  
Zijian Fang ◽  
Stavroula Bitsi ◽  
Johannes Broichhagen ◽  
...  
Keyword(s):  
Blood Glucose ◽  
Cell Types ◽  
Cellular Level ◽  
Control Blood Glucose ◽  
C Terminus ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
The Impact

AbstractBackground and purposeAmino acid substitutions at the N-termini of glucagon-like peptide-1 receptor agonist (GLP-1RA) peptides result in distinct patterns of intracellular signalling, sub-cellular trafficking and efficacy in vivo. Here we aimed to determine whether sequence differences at the ligand C-termini of clinically approved GLP-1RAs exendin-4 and lixisenatide lead to similar phenomena. We also sought to establish the impact of the C-terminus on signal bias resulting from modifications elsewhere in the molecule.Experimental approachExendin-4, lixisenatide, and N-terminally substituted analogues with biased signalling characteristics were compared across a range of in vitro trafficking and signalling assays in different cell types. Fluorescent ligands and new time-resolved FRET approaches were developed to study agonist behaviours at the cellular and sub-cellular level. Anti-hyperglycaemic and anorectic effects of each parent ligand, and their biased derivatives, were assessed in mice.Key resultsLixisenatide and exendin-4 showed equal binding affinity, but lixisenatide was 5-fold less potent for cAMP signalling. Both peptides were rapidly endocytosed, but the GLP-1R recycled more slowly to the plasma membrane after lixisenatide treatment. These combined deficits resulted in reduced maximal sustained insulin secretion and reduced anti-hyperglycaemic and anorectic effects in mice. N-terminal substitutions to both ligands had favourable effects on their pharmacology, resulting in improved insulin release and lowering of blood glucose.Conclusion and implicationsChanges to the C-terminus of exendin-4 affect signalling potency and GLP-1R trafficking via mechanisms unrelated to GLP-1R occupancy. These differences were associated with changes in their ability to control blood glucose and therefore may be therapeutically relevant.

scholarly journals Acute Mechanical Stress in Primary Porcine RPE Cells Induces Angiogenic Factor Expression and in vitro Angiogenesis

2020 ◽  
Author(s):  
Farhad Farjood ◽  
Amir Ahmadpour ◽  
Sassan Ostvar ◽  
Elizabeth Vargis
Keyword(s):  
Mechanical Stress ◽  
Tube Formation ◽  
Angiogenic Factors ◽  
Angiogenic Factor ◽  
Rpe Cells ◽  
Qrt Pcr ◽  
Angiogenic Proteins ◽  
The Impact ◽  
In Vitro Angiogenesis

Abstract Background Choroidal neovascularization (CNV) is a major cause of blindness in patients with age-related macular degeneration. CNV is characterized by new blood vessel growth and subretinal fluid accumulation, which results in mechanical pressure on retinal pigment epithelial (RPE) cells. The overexpression of RPE-derived angiogenic factors plays an important role in inducing CNV. In this work, we investigated the effect of mechanical stress on the expression of angiogenic factors in porcine RPE cells and determined the impact of conditioned medium on in-vitro angiogenesis. Results The goal of this study was to determine whether low levels of acute mechanical stress during early CNV can induce the expression of angiogenic factors in RPE cells and accelerate angiogenesis. Using a novel device, acute mechanical stress was applied to primary porcine RPE cells and the resulting changes in the expression of major angiogenic factors, VEGF, ANG2, HIF-1α, IL6, IL8 and TNF - α, were examined using immunocytochemistry, qRT-PCR, and ELISA. An in vitro tube formation assay was used to determine the effect of secreted angiogenic proteins due to mechanical stress on endothelial tube formation by human umbilical vein endothelial cells (HUVECs). Our results showed an increase in the expression of VEGF, ANG2, IL-6 and IL-8 in response to mechanical stress, resulting in increased in vitro angiogenesis. Abnormal epithelial-mesenchymal transition (EMT) in RPE cells is also associated with CNV and further retinal degeneration. Our qRT-PCR results verified an increase in the expression of EMT genes, CDH2, VIM and FN1, in RPE cells. Conclusions In conclusion, we showed that acute mechanical stress induces the expression of major angiogenic and EMT factors and promotes in vitro angiogenesis, suggesting that mechanical stress plays a role in promoting aberrant angiogenesis in AMD.

Synthesis and in vitro biological evaluation of new pyrimidines as glucagon-like peptide-1 receptor agonists

2017 ◽  
Vol 27 (22) ◽  
pp. 5071-5075 ◽  
Author(s):  
Shaikha S. AlNeyadi ◽  
Abdu Adem ◽  
Naheed Amer ◽  
Alaa A. Salem ◽  
Ibrahim M. Abdou
Keyword(s):  
Biological Evaluation ◽  
Receptor Agonists ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1

scholarly journals A synthetic glucagon-like peptide-1 analog with improved plasma stability

1998 ◽  
Vol 159 (1) ◽  
pp. 93-102 ◽  
Author(s):  
U Ritzel ◽  
U Leonhardt ◽  
M Ottleben ◽  
A Ruhmann ◽  
K Eckart ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plasma Insulin ◽  
Rat Plasma ◽  
Plasma Stability ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1

Glucagon-like peptide-1 (GLP-1) is the most potent endogenous insulin-stimulating hormone. In the present study the plasma stability and biological activity of a GLP-1 analog, [Ser]GLP-1(7-36)amide, in which the second N-terminal amino acid alanine was replaced by serine, was evaluated in vitro and in vivo. Incubation of GLP-1 with human or rat plasma resulted in degradation of native GLP-1(7-36)amide to GLP-1(9-36)amide, while [Ser]GLP-1(7-36)amide was not significantly degraded by plasma enzymes. Using glucose-responsive HIT-T15 cells, [Ser]GLP-1(7-36)amide showed strong insulinotropic activity, which was inhibited by the specific GLP-1 receptor antagonist exendin-4(9-39)amide. Simultaneous i.v. injection of [Ser]GLP-1(7-36)amide and glucose in rats induced a twofold higher increase in plasma insulin levels than unmodified GLP-1(7-36)amide with glucose and a fivefold higher increase than glucose alone. [Ser]GLP-1(7-36)amide induced a 1.5-fold higher increase in plasma insulin than GLP-1(7-36)amide when given 1 h before i.v. application of glucose. The insulinotropic effect of [Ser]GLP-1(7-36)amide was suppressed by i.v. application of exendin-4(9-39)amide. The present data demonstrate that replacement of the second N-terminal amino acid alanine by serine improves the plasma stability of GLP-1(7-36)amide. The insulinotropic action in vitro and in vivo was not impaired significantly by this modification.

scholarly journals Cyclic AMP-dependent protein kinase regulates transcription of the phosphoenolpyruvate carboxykinase gene but not binding of nuclear factors to the cyclic AMP regulatory element.

1990 ◽  
Vol 10 (7) ◽  
pp. 3357-3364 ◽  
Author(s):  
P G Quinn ◽  
D K Granner
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Rat Liver ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Nuclear Extract ◽  
In Vitro Transcription ◽  
Catalytic Subunit ◽  
Nuclear Factors ◽  
Dependent Protein

We have examined the binding of factors in rat liver nuclear extracts to the phosphoenolpyruvate carboxykinase (PEPCK) gene cyclic AMP (cAMP) response element (CRE) and other CREs and have isolated a rat liver CRE-binding protein (CREBP) cDNA. In addition, we have examined the influence of altering the phosphorylation state of nuclear factors on both CRE binding and in vitro transcription. Specific binding to the PEPCK CRE was measured in a mobility shift assay. CRE sequences of the PEPCK, somatostatin, and glycoprotein hormone alpha subunit genes competed equally for binding of rat liver nuclear factors to the PEPCK CRE, whereas mutant PEPCK CRE sequences did not compete for binding. Oligonucleotides complementary to rat pheochromocytoma CREBP (Gonzalez et al., Nature [London] 337:749-752, 1989) were used to prime rat liver and brain cDNA in the polymerase chain reaction. The predominant CREBP molecule obtained was identical to the rat pheochromocytoma CREBP except for a 14-amino-acid deletion in the N-terminal half that was also present in a human placental cDNA (Hoeffler et al., Science 242:1430-1433, 1988). The regulation of transcription by cAMP was examined by coincubation of rat liver nuclear extract with the purified catalytic subunit of cAMP-dependent protein kinase (protein kinase A). Although binding to the CRE was unaffected, in vitro transcription directed by the PEPCK promoter was stimulated by catalytic subunit, and this effect was blocked by protein kinase inhibitor peptide. In contrast, when nuclear extract was coincubated with phosphatase, there was substantial inhibition of in vitro transcription directed by the PEPCK promoter, but there was no effect on binding to the CRE. The major effects of catalytic subunit were exerted through the CRE, but residual stimulation was evident in promoter fragments containing only the TATA element. These data suggest that factors are bound to the CRE at constitutively high levels and that their capacity for transcriptional activation is regulated by phosphorylation.

In Vitro Metabolism of the Glucagon-Like Peptide-1 (GLP-1)–Derived Metabolites GLP-1(9-36)amide and GLP-1(28-36)amide in Mouse and Human Hepatocytes

2013 ◽  
Vol 41 (12) ◽  
pp. 2148-2157 ◽  
Author(s):  
Raman Sharma ◽  
Thomas S. McDonald ◽  
Heather Eng ◽  
Chris Limberakis ◽  
Benjamin D. Stevens ◽  
...  
Keyword(s):  
Human Hepatocytes ◽  
In Vitro Metabolism ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1

Abstract 3: Il-10 Regulated Mir-375 Enhances Endothelial Progenitor Cell Mediated Myocardial Repair And Survival After Myocardial Infarction.

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Venkata N Garikipati ◽  
Prasanna Krishnamurthy ◽  
Suresh K Verma ◽  
Alexandra R Mackie ◽  
Erin E Vaughan ◽  
...  
Keyword(s):  
Capillary Density ◽  
Tube Formation ◽  
Lv Function ◽  
Myocardial Repair ◽  
Knock Down ◽  
Cardiac Functions ◽  
Dependent Protein ◽  
And Function ◽  
Deficient Mice

We hypothesized that IL-10 regulates miR-375 signaling in EPCs to enhance their survival and function in ischemic myocardium after MI. miR-375 knock down EPC were transplanted intramyocardially after induction of MI. Mice receiving EPC treated with miR-375 inhibitor showed increased number of GFP+EPCs retention that was associated with reduced EPC apoptosis in the myocardium. The engraftment of EPC into the vascular structures and the associated capillary density was significantly higher in miR-375-treated mice. The above findings further correlated with reduced infarct size, fibrosis and enhanced LV function (echocardiography) in miR-375 knock down EPC group as compared to scrambled EPC. Our in vitro studies revealed that the knockdown of miR-375 enhanced EPC proliferation, migration; tube formation ability and inhibited cell apoptosis, while the up-regulation of miR-375 with the mimic had the opposite effects. In addition, we found that miR-375 negatively regulates the expression of 3-phosphoinositide-dependent protein kinase 1 (PDK1) by directly targeting the 3'UTR of the PDK1 transcript. Interestingly, EPC isolated from IL-10-deficient mice has elevated basal levels of miR-375 and exhibited poor proliferation and tube formation ability where as miR-375 knock down in EPC isolated from IL-10 deficient mice attenuated these effects. Furthermore, transplantation of miR-375 knock down IL-10 deficient EPC after MI resulted in attenuated cardiac functions compared to scramble IL-10 deficient EPCs. Taken together, our studies suggest that IL-10 regulated miR-375 enhances EPC survival and function, associated with efficient myocardial repair via activation of PDK-1/AKT signaling cascades.

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