Effects Of Ex-4 Research Articles

Reduction of hippocampal IL-6 levels in LPS-injected rats following acute exendin-4 treatment.

Preclinical evidence on the role of glucagon-like peptide-1 receptor (GLP-1r) agonists in the brain led to an increased interest in repurposing these compounds as a therapy for central nervous system (CNS) disorders and associated comorbidities. We aimed to investigate the neuroprotective effects of acute treatment with exendin (EX)-4, a GLP-1r agonist, in an animal model of inflammation. We evaluated the effect of different doses of EX-4 on inflammatory, neurotrophic, and oxidative stress parameters in the hippocampus and serum of lipopolysaccharide (LPS)-injected animals. Male Wistar rats were injected with LPS (0.25mg/kgi.p.) and treated with different doses of EX-4 (0.1, 0.3, or 0.5μg/kgi.p.). Sickness behavior was assessed by locomotor activity and body weight, and depressive-like behavior was also evaluated using forced swim test (FST). Brain-derived neurotrophic factor (BDNF), thiobarbituric acid reactive species (TBARS), and interleukin (IL)-6 were quantified in the serum and hippocampus. Glycemia was also analyzed pre- and post-EX-4 treatment. LPS groups exhibited decreased frequency of crossing and reduced body weight (p < 0.001), while alterations on FST were not observed. The higher dose of EX-4 reduced IL-6 in the hippocampus of LPS-injected animals (p = 0.018), and EX-4 per se reduced TBARS serum levels with a modest antioxidant effect in the LPS groups (p ≤ 0.005). BDNF hippocampal levels seemed to be increased in the LPS+EX-4 0.5 group compared with LPS+Saline (p > 0.05). Our study provides evidence on acute anti-inflammatory effects of EX-4 in the hippocampus of rats injected with LPS, contributing to future studies on repurposing compounds with potential neuroprotective properties.

The glucagon-like peptide-1 receptor agonist reduces inflammation and blood-brain barrier breakdown in an astrocyte-dependent manner in experimental stroke

BackgroundPreserving the integrity of the blood-brain barrier (BBB) is beneficial to avoid further brain damage after acute ischemic stroke (AIS). Astrocytes, an important component of the BBB, promote BBB breakdown in subjects with AIS by secreting inflammatory factors. The glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 (Ex-4) protects the BBB and reduces brain inflammation from cerebral ischemia, and GLP-1R is expressed on astrocytes. However, the effect of Ex-4 on astrocytes in subjects with AIS remains unclear.MethodsIn the present study, we investigated the effect of Ex-4 on astrocytes cultured under oxygen-glucose deprivation (OGD) plus reoxygenation conditions and determined whether the effect influences bEnd.3 cells. We used various methods, including permeability assays, western blotting, immunofluorescence staining, and gelatin zymography, in vitro and in vivo.ResultsEx-4 reduced OGD-induced astrocyte-derived vascular endothelial growth factor (VEGF-A), matrix metalloproteinase-9 (MMP-9), chemokine monocyte chemoattractant protein-1 (MCP-1), and chemokine C-X-C motif ligand 1 (CXCL-1). The reduction in astrocyte-derived VEGF-A and MMP-9 was related to the increased expression of tight junction proteins (TJPs) in bEnd.3 cells. Ex-4 improved neurologic deficit scores, reduced the infarct area, and ameliorated BBB breakdown as well as decreased astrocyte-derived VEGF-A, MMP-9, CXCL-1, and MCP-1 levels in ischemic brain tissues from rats subjected to middle cerebral artery occlusion. Ex-4 reduced the activation of the JAK2/STAT3 signaling pathway in astrocytes following OGD.ConclusionBased on these findings, ischemia-induced inflammation and BBB breakdown can be improved by Ex-4 through an astrocyte-dependent manner.

Exendin-4 regulates Wnt and NF-κB signaling in lipopolysaccharide-induced human periodontal ligament stem cells to promote osteogenic differentiation

A major feature of chronic periodontitis (CP) is the damage and destruction of alveolar bone. Periodontal ligament stem cells (PDLSCs) can differentiate into bone and improve CP. Exendin-4 (Ex-4) has been shown to have anti-inflammatory mechanisms and can promote bone regeneration. However, the effects of Ex-4 on the osteogenic differentiation of PDLSCs in the inflammatory microenvironment remains uncharacterized. In this study, we assessed the effects of Ex-4 on PDLSCs stimulated with lipopolysaccharide (LPS) to mimic the inflammatory environment. PDLSCs proliferation was assessed through CCK-8 assays and osteogenic differentiation was measured using Alizarin Red staining. The anti-inflammatory and osteogenic mechanisms of Ex-4 were assessed by western blot, RT-PCR, ELISA and immunofluorescence. We found that LPS treatment promoted the proliferative capacity of PDLSCs and inhibited their osteogenic differentiation. However, Ex-4 reversed these effects through suppressing PDLSCs proliferation and promoting osteogenic differentiation. Ex-4 increased Runx2, ALP, and Osx levels and decreased TNF-α and IL-6 expression. Ex-4 also reduced the expression of IκBα and p-IκBα, and inhibited the nuclear translocation of NF-κB/p65. The expression of β-catenin decreased in nucleus after co-treatment of Ex-4 with LPS. Taken together, these data demonstrate that Ex-4 promotes PDLSCs osteogenic differentiation in the inflammatory microenvironment through regulating NF-κB and Wnt signaling.

1954-P: Combined Treatment with Exendin-4 and Metformin Attenuates Breast Cancer Growth

Cancer is a major cause of death in patients with diabetes. Incretin therapy has received much attention, because of its tissue protective effects beyond glycemic control. We have previously reported anti-prostate cancer effect of Ex-4 and metformin (Diabetes 2014, PLOS ONE 2015). In addition, we have recently reported that Ex-4 attenuates breast cancer growth (Endocrinology 2017). Then, we next examined the effect of combined treatment of Ex-4 with Metformin in breast cancer. In human breast cancer cell lines, MCF-7, MDA-MB-231 and KPL-1 cells, Ex-4 or metformin reduced cell number in a dose-dependent manner. Further, combined treatment of Metformin and Ex-4 additively attenuated the growth curve progression of breast cancer cells. In BrdU assay, Ex-4 or Metformin significantly decreased breast cancer cell proliferation, and further reduction of BrdU incorporation was observed by combined treatment with Ex-4 and Metformin, suggesting that Ex-4 and Metformin additively decreased DNA synthesis in breast cancer cells. Although apoptotic cells were not observed in Ex-4 treated breast cancer cells, apoptotic cells were clearly detected in metformin treated breast cancer cells in TUNEL assay. Further, the experiments using xenograft model mice transplanting MCF-7 cells into athymic mice revealed that Ex-4 and metformin additively attenuated breast cancer growth in vivo also. These data suggest that Ex-4 and metformin attenuates breast cancer cell proliferation and metformin induces apoptosis in breast cancer cells and combined treatment with Ex-4 and metformin could be an optional therapy to inhibit breast cancer progression. Disclosure Y. Fujimura: None. T. Nomiyama: None. C. Iwaya: None. T. Kawanami: None. Y. Hamaguchi: None. T. Tanaka: None. T. Yanase: None.

Effects of Exendin-4 on the differentiation of neural stem cells from subventricular zone of adult mice in vitro

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 . 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. 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). 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.

PI 3-kinase- and ERK-MAPK-dependent mechanisms underlie Glucagon-Like Peptide-1-mediated activation of Sprague Dawley colonic myenteric neurons.

Glucagon-like peptide (GLP-1) can modify colonic function, with beneficial effects reported in the functional bowel disorder, irritable bowel syndrome (IBS). IBS pathophysiology is characterized by hyper-activation of the hypothalamic-pituitary-adrenal stress axis and altered microbial profiles. This study aims to characterize the neuronal and functional effects of GLP-1 in healthy rat colons to aid understanding of its beneficial effects in moderating bowel dysfunction. Immunofluorescent and calcium imaging of myenteric neurons prepared from Sprague Dawley rat colons was carried out to elucidate the neuromodulatory actions of the GLP-1 receptor agonist, exendin-4 (Ex-4). Colonic contractile activity was assessed using organ bath physiological recordings. Ex-4 induced an elevation of intracellular calcium arising from store release and influx via voltage-gated calcium channels. Ex-4 activated both ERK-MAPK and PI 3-kinase signaling cascades. Neuronal activation was found to underlie suppression of contractile activity in colonic circular muscle. Although the stress hormone, corticotropin-releasing factor (CRF) potentiated the neuronal response to Ex-4, and the functional effects of Ex-4 on colonic circular muscle activity were not altered. Ex-4 evoked neurally regulated suppression of rat colonic circular muscle activity. In myenteric neurons, the neurostimulatory effects of Ex-4 were dependent upon activation of PI 3-kinase and ERK-MAPK signaling cascades. No further change in circular muscle function was noted in the presence of CRF suggesting that stress does not impact on colonic function in health. Further studies in a model of IBS are needed to determine whether mechanisms are modified in the context of bowel dysfunction.

PYY(3-36) and exendin-4 reduce food intake and activate neuronal circuits in a synergistic manner in mice

Peptide YY(3-36) ((PYY(3-36)) and glucagon like peptide 1 (GLP-1) in combination reduce food intake and body weight in an additive or synergistic manner in animal models and in humans. Nevertheless, the mechanisms behind are not completely understood. The present study aims to investigate the effect of combining PYY(3-36) and the GLP-1 receptor agonist exendin-4 (Ex4) by examining acute food intake and global neuronal activation as measured by c-fos in C57BL/6 J mice. An additive reduction in food intake was found 1.5 h after s.c dosing with the combination of PYY(3-36) (200 μg/kg) and Ex4 (2.5 μg/kg). This was associated with a synergistic enhancement of c-fos reactivity in central amygdalar nucleus (CeA), rostral part of the mediobasal arcuate nucleus (ARH), supratrigeminal nucleus (SUT), lateral parabrachial nucleus (PB), area postrema (AP) and nucleus tractus solitarius (NTS) compared to vehicle, PYY(3-36) and Ex4 individually dosed mice. The regions activated by Ex4 individually and PYY(3-36) and Ex4 in combination resembled each other, but the combination group had a significantly stronger c-fos response. Twenty-five brain areas were activated by PYY(3-36) and Ex4 in combination compared to vehicle versus nine brain areas by Ex4 individually. No significant increase in c-fos reactivity was found by PYY(3-36) compared to vehicle dosed mice. The neuronal activation of ARH and the AP/NTS to PB to CeA pathway is important for appetite regulation while SUT has not previously been reported in the regulation of energy balance. As PYY(3-36) and Ex4 act on different neurons leading to recruitment of different signalling pathways within and to the brain, an interaction of these pathways may contribute to their additive/synergistic action. Thus, PYY(3-36) boosts the effect of Ex4 possibly by inducing less inhibition of neuronal activity leading to an enhanced neuronal activity induced by Ex4.

Modulation of Cardiac Ventricular Excitability by GLP-1 (Glucagon-Like Peptide-1).

BackgroundGlucagon-like peptide-1 receptor (GLP-1R) agonists improve cardiovascular outcomes in patients with type 2 diabetes mellitus. However, systemic actions of these agents cause sympathetic activation, which is generally considered to be detrimental in cardiovascular disease. Despite significant research interest in cardiovascular biology of GLP-1, the presence of GLP-1R in ventricular cardiomyocytes remains a controversial issue, and the effects of this peptide on the electrical properties of intact ventricular myocardium are unknown. We sought to determine the effects of GLP-1R agonist exendin-4 (Ex4) on ventricular action potential duration (APD) and susceptibility to ventricular arrhythmia in the rat heart in vivo and ex vivo.MethodsVentricular monophasic action potentials were recorded in anaesthetized (urethane) rats in vivo and isolated perfused rat hearts during sinus rhythm and ventricular pacing.ResultsIn vivo, systemic administration of Ex4 (5 μg/kg intravenously) increased heart rate, and this effect was abolished by β-adrenoceptor blockade. Despite causing sympathetic activation, Ex4 increased APD at 90% repolarization during ventricular pacing by 7% (P=0.044; n=6) and reversed the effect of β-adrenoceptor agonist dobutamine on APD at 90% repolarization. In isolated perfused hearts, Ex4 (3 nmol/L) increased APD at 90% repolarization by 14% (P=0.015; n=6) with no effect on heart rate. Ex4 also reduced ventricular arrhythmia inducibility in conditions of β-adrenoceptor stimulation with isoproterenol. Ex4 effects on APD and ventricular arrhythmia susceptibility were prevented in conditions of muscarinic receptor blockade or inhibition of nitric oxide synthase.ConclusionsThese data demonstrate that GLP-1R activation effectively opposes the effects of β-adrenoceptor stimulation on cardiac ventricular excitability and reduces ventricular arrhythmic potential. The effect of GLP-1R activation on the ventricular myocardium is indirect, mediated by acetylcholine and nitric oxide and, therefore, can be explained by stimulation of cardiac parasympathetic (vagal) neurons.

Activation of GLP-1 receptor signalling alleviates cellular stresses and improves beta cell function in a mouse model of Wolfram syndrome.

Loss of functional beta cells results in a gradual progression of insulin insufficiency in Wolfram syndrome caused by recessive WFS1 mutations. However, beta cell dysfunction in Wolfram syndrome has yet to be fully characterised, and there are also no specific treatment recommendations. In this study, we aimed to characterise beta cell secretory defects and to examine the potential effects of a glucagon-like peptide-1 (GLP-1) receptor agonist on diabetes in Wolfram syndrome. Insulin secretory function was assessed by the pancreatic perfusion method in mice used as a model of Wolfram syndrome. In addition, granule dynamics in living beta cells were examined using total internal reflection fluorescence microscopy. Acute and chronic effects of exendin-4 (Ex-4) on glucose tolerance and insulin secretion were examined in young Wfs1-/- mice without hyperglycaemia. Molecular events associated with Ex-4 treatment were investigated using pancreatic sections and isolated islets. In addition, we retrospectively observed a woman with Wolfram syndrome who had been treated with liraglutide for 24weeks. Treatment with liraglutide ameliorated our patient's glycaemic control and resulted in a 20% reduction of daily insulin dose along with an off-drug elevation of fasting C-peptide immunoreactivity. Glucose-stimulated first-phase insulin secretion and potassium-stimulated insulin secretion decreased by 53% and 59%, respectively, in perfused pancreases of 10-week-old Wfs1-/- mice compared with wild-type (WT) mice. The number of insulin granule fusion events in the first phase decreased by 41% in Wfs1-/- beta cells compared with WT beta cells. Perfusion with Ex-4 increased insulin release in the first and second phases by 3.9-fold and 5.6-fold, respectively, in Wfs1-/- mice compared with perfusion with saline as a control. The physiological relevance of the effects of Ex-4 was shown by the fact that a single administration potentiated glucose-stimulated insulin secretion and improved glucose tolerance in Wfs1-/- mice. Four weeks of administration of Ex-4 resulted in an off-drug amelioration of glucose excursions after glucose loading in Wfs1-/- mice, with insulin secretory dynamics that were indistinguishable from those in WT mice, despite the fact that there was no alteration in beta cell mass. In association with the functional improvements, Ex-4 treatment reversed the increases in phosphorylated eukaryotic initiation factor (EIF2α) and thioredoxin interacting protein (TXNIP), and the decrease in phosphorylated AMP-activated kinase (AMPK), in the beta cells of the Wfs1-/- mice. Furthermore, Ex-4 treatment modulated the transcription of oxidative and endoplasmic reticulum stress-related markers in isolated islets, implying that it was able to mitigate the cellular stresses resulting from Wfs1 deficiency. Our study provides deeper insights into the pathophysiology of beta cell dysfunction caused by WFS1 deficiency and implies that activation of the GLP-1 receptor signal may alleviate insulin insufficiency and aid glycaemic control in Wolfram syndrome.

Exendin-4, a Glucagon-Like Peptide-1 Receptor Agonist, Attenuates Prostate Cancer Cell Proliferation via Phosphorylation of MKP-1

Pleiotropic benefits of incretin therapy beyond glycemic control have been reported and receiving much attention. Cancer is one of major causes of death in patients with type 2 diabetes. We have previously reported Exendin-4(Ex-4), a glucagon-like peptide-1 receptor agonist, attenuates prostate cancer growth by the inhibition of ERK-MAPK phosphorylation (Diabetes 2014). On the other hand, it is well known that MAPK is inactivated and tightly regulated by MAPK phosphatase (MKP) family. Then, we next examined whether MKP could be involved in the anti-proliferative effect of Ex-4 in the prostate cancer cell. In a human prostate cancer cell line, LNCaP cell, MKP-1 was abundantly expressed. Knocking down of MKP-1 using siRNA abolished Ex-4 induced-reduction of LNCaP cell number in growth curve. In addition, Ex-4 treatment significantly increased MKP-1 mRNA expression. To activate its phosphatase activity, Ser359 in the C terminus of MKP-1 is specifically phosphorylated. Ex-4 significantly phosphorylated Ser359 of MKP-1along with time course of dephosphorylating ERK-MAPK. Using Immunoprecipitation assay, we further confirmed that Ex-4 exactly phosphorylated Ser359 of MKP-1. In addition, if we transiently transfected GFP conjugated MKP-1 into LNCaP cells, nuclear translocation of MKP-1 induced by Ex-4 treatment was observed in early phase stimulation. These data suggest that Ex-4 attenuates prostate cancer cell proliferation via phosphorylation of MKP-1 followed by inhibition of ERK-MAPK activation. Disclosure T. Kawanami: None. T. Nomiyama: Research Support; Self; MSD K.K., Sanofi-Aventis, Boehringer Ingelheim Pharmaceuticals, Inc., Takeda Pharmaceuticals, Japan. Y. Hamaguchi: None. T. Tanaka: None. T. Yanase: Research Support; Self; MSD K.K., Boehringer Ingelheim Pharmaceuticals, Inc., Takeda Pharmaceuticals, Japan.

Combined Treatment with Exendin-4 and Metformin Attenuates Breast Cancer Growth

Cancer is a major cause of death in patients with diabetes. Incretin therapy has received much attention, because of its tissue protective effects beyond glycemic control. We have previously reported anti-prostate cancer effect of Ex-4 and metformin (Diabetes 2014, PLOS ONE 2015). In addition, we have recently reported that Ex-4 attenuates breast cancer growth (Endocrinology 2017). Then, we next examined the effect of combined treatment of Ex-4 with Metformin in breast cancer cells. In human breast cancer cell lines, MCF-7, MDA-MB-231 and KPL-1 cell, Ex-4 or metformin reduced cell number in a dose-dependent manner. Further, combined treatment of 0.1mM Metformin and 10nM Ex-4 additively attenuated the growth curve progression of breast cancer cells. In BrdU assay, Ex-4 or Metformin significantly decreased breast cancer cell proliferation, and further reduction of BrdU incorporation was observed by combined treatment of Ex-4 and Metformin, suggesting that Ex-4 and Metformin additively decreased DNA synthesis in breast cancer cells. Although apoptotic cells were not observed in Ex-4 treated breast cancer cells similar to our previous study, apoptotic cells were clearly detected in metformin treated breast cancer cells using TUNEL assay. These data suggest that Ex-4 and metformin attenuates breast cancer cell proliferation and metformin induces apoptosis in breast cancer cells. Combined treatment of Ex-4 and Metformin could be an optional therapy to inhibit breast cancer progression. Disclosure C. Iwaya: None. T. Nomiyama: Research Support; Self; MSD K.K., Sanofi-Aventis, Boehringer Ingelheim Pharmaceuticals, Inc., Takeda Pharmaceuticals, Japan. T. Kawanami: None. Y. Hamaguchi: None. T. Tanaka: None. T. Yanase: Research Support; Self; MSD K.K., Boehringer Ingelheim Pharmaceuticals, Inc., Takeda Pharmaceuticals, Japan.

Glucagon-like peptide-1 regulates brown adipose tissue thermogenesis via the gut-brain axis in rats.

Endogenous intestinal glucagon-like peptide-1 (GLP-1) controls satiation and glucose metabolism via vagal afferent neurons (VANs). Recently, VANs have received increasing attention for their role in brown adipose tissue (BAT) thermogenesis. It is, however, unclear whether VAN GLP-1 receptor (GLP-1R) signaling affects BAT thermogenesis and energy expenditure (EE) and whether this VAN mechanism contributes to energy balance. First, we tested the effect of the GLP-1R agonist exendin-4 (Ex4, 0.3 μg/kg ip) on EE and BAT thermogenesis and whether these effects require VAN GLP-1R signaling using a rat model with a selective Glp1r knockdown (kd) in VANs. Second, we examined the role of VAN GLP-1R in energy balance during chronic high-fat diet (HFD) feeding in VAN Glp1r kd rats. Finally, we used viral transsynaptic tracers to identify the possible neuronal substrates of such a gut-BAT interaction. VAN Glp1r kd attenuated the acute suppressive effects of Ex4 on EE and BAT thermogenesis. Consistent with this finding, the VAN Glp1r kd increased EE and BAT activity, diminished body weight gain, and improved insulin sensitivity compared with HFD-fed controls. Anterograde transsynaptic viral tracing of VANs infected major hypothalamic and hindbrain areas involved in BAT sympathetic regulation. Moreover, retrograde tracing from BAT combined with laser capture microdissection revealed that a population of VANs expressing Glp1r is synaptically connected to the BAT. Our findings reveal a novel role of VAN GLP-1R signaling in the regulation of EE and BAT thermogenesis and imply that through this gut-brain-BAT connection, intestinal GLP-1 plays a role in HFD-induced metabolic syndrome.

Central Fructose Impairs Exendin 4‐Mediated Anorexia in the Ventromedial Hypothalamus in Male C57Bl/6 Mice

Glucagon‐like peptide‐1 (Glp1) is a gut hormone that stimulates insulin secretion but also promotes satiety via actions on the central nervous system. As such, Glp1 receptor (Glp1r) agonists are currently being developed as therapeutics for body weight control. We have previously shown that pharmacological activation of the Glp1r in the ventromedial hypothalamus (VMH) by the Glp1r agonist Exendin‐4 (Ex4) suppresses food intake via stimulation of intracellular glucose metabolism and inhibition of the cellular nutrient sensor AMP‐activated protein kinase (AMPK). This raises the possibility that interference with these nutrient‐sensing mechanisms may attenuate the anorectic effect of Glp1r agonists in the brain. The dietary monosaccharide fructose is an AMPK activator, and intracerebroventricular (ICV) fructose attenuates the anorectic effect of ICV Ex4. The relevance of this finding is underscored by evidence supporting an association between excessive dietary fructose consumption (e.g., high fructose corn syrup, sucrose) and the obesity epidemic. The aim of these studies was to assess whether fructose also impairs Ex4‐mediated anorexia in the VMH. Male C57Bl/6 mice were maintained on a chow diet and were instrumented with bilateral cannulae targeting the VMH for delivery of Ex4 following (1) VMH‐targeted or (2) orally‐gavaged pre‐treatment with fructose, glucose or vehicle. In additional studies, animals were challenged with a high fructose (vs. carbohydrate control) diet for 15wks prior to treatment with Ex4 in the VMH. 18‐h food intake, energy expenditure (EE) and locomotor activity were assessed using a Comprehensive Lab Animal Monitoring System. VMH‐targeted fructose significantly attenuated the food intake‐suppressive effect of VMH Ex4 but had no effect on EE. VMH‐targeted glucose, however, did not potentiate the anorectic effect of Ex4. Orally‐dosed fructose did not impair the anorectic effect of Ex4 in the VMH, nor did dietary fructose. Similarly, orally‐dosed glucose did not potentiate the anorectic effect of Ex4 in the VMH. In conclusion, although fructose impairs the ability of Ex4 to reduce food intake when targeted to the VMH, it is not an inhibitor of VMH Glp1r action on food intake when it is administered along an ingested route (i.e., either orally or dietary). However, these observations validate the role of AMPK as a mediator of the anorectic effect of Ex4 in the VMH. Further elucidation of the interactions between fructose and Glp1r‐mediated signaling in the brain could be beneficial in developing more effective anti‐obesity therapies.Support or Funding InformationThis work was supported by NIH R01 1R01DK097361 to Julio AyalaThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of glucagon-like peptide-1 analogue; Exendin-4, on cognitive functions in type 2 diabetes mellitus; possible modulation of brain derived neurotrophic factor and brain Visfatin

BackgroundBrain derived neurotrophic factor (BDNF) is one of the most essential neurotrophic factors in the brain. BDNF is involved in learning, memory and locomotion suggesting it as a target in type 2 diabetes mellitus (T2DM) associated cognitive changes. Visfatin; an adipokine discovered to be expressed in the brain; was found to have multiple effects including its participation in keeping energy supply to the cell and is consequentially involved in cell survival. Its role in cognitive functions in T2DM was not studied before. Recent studies point to the possible neuro-protective mechanisms of glucagon-like peptide 1 analogue: Exendin-4 (Ex-4) in many cognitive disorders, but whether BDNF or Visfatin are involved or not in its neuro-protective mechanisms; is still unknown. Aimsto study the changes in cognitive functions in T2DM, either not treated or treated with Glucagon-like peptide 1 (GLP-1) analogue: Ex-4, and to identify the possible underlying mechanisms of these changes and whether BDNF and brain Visfatin are involved. MethodsA total of 36 adult male wistar albino rats were divided into 4 groups; Control, Exendin-4 control, Diabetic and Exendin-4 treated groups. At the end of the study, Y-maze and open field tests were done the day before scarification to assess spatial working memory and locomotion, respectively. Fasting glucose and insulin, lipid profile and tumor necrosis factor- alpha (TNF-α) were measured in the serum. Homeostasis model assessment insulin resistance was calculated. In the brain tissue, malondialdehyde (MDA) level, gene expression and protein levels of BDNF and Visfatin, area of degenerated neurons, area of glial cells and area % of synaptophysin immunoexpression were assessed. ResultsCompared with the control, the untreated diabetic rats showed insulin resistance, dyslipidemia and elevation of serum TNF-α. The brain tissue showed down-regulation of BDNF gene expression and reduction of its protein level, up-regulation of Visfatin gene expression and elevation of its protein level, increase in MDA, area of degenerated neurons and area of glial cells and reduction in area % of synaptophysin immunoexpression. These changes were paralleled with significant deterioration in spatial working memory and locomotion. Treatment of diabetic rats with Ex-4 reversed all these changes. ConclusionT2DM has a negative impact on cognitive functions through different pathological and subcellular mechanisms. The current study provides evidence for involvement of BDNF and brain Visfatin in T2DM- associated cognitive dysfunction. BDNF and brain Visfatin were also found to contribute to the neuro-protective effect of Ex-4 via modulation of inflammation, oxidative stress, neuro-degeneration and synaptic function.

Exendin-4 dose not evoke pancreatitis or pancreatitis-associated histopathological and genetic changes in high-fat diet induced diabetic mice

Background: Clinical reports have suggested the potential link of Glucagon-Like Peptide 1 Receptor Agonists (GLP-1RAs) with the development of pancreatitis. We investigated the effects of long-term exposure of Exendin-4 (Ex-4), a kind of GLP-1RA, on biochemical, histological and genetic markers of pancreatitis in High-Fat Diet (HFD) induced mice. Methodology: HFD induced mice received subcutaneous twice-daily injections of Ex-4 (3 and 30 μg/kg/d) or vehicle for 12 w. Pancreatitis was induced with Caerulein (CRN) in Ex-4 treated mice or wild mice. Changes in body weight, food consumption, serum amylase, serum lipase, glucose, and insulin concentrations were measured in each group. An extended histopathological and ultralstructural by transmission electron microscopy evaluation of exocrine pancreas was performed. The expressions of pancreatitis-associated microRNAs and genes were assessed by real-time PCR. Results: Ex-4 improved physical condition, glucose concentrations, decreased food intake, and increased serum insulin sensitivity in HFD induced mice. No deleterious effects on serum amylase and lipase were observed after Ex-4 treatment. The histopathological and ultralstructural findings did not reveal adverse effects of Ex-4. Ex-4 administration did not significantly modify the level of pancreatitisassociated microRNAs (miR-181, miR-148, miR-210 and miR-216a) or pancreatitis-associated genes (RegIII, MCP-1 and IL-6). However, we noted slightly pancreatitis ultralstructural changes in one mouse from 30 μg/kg/d Ex-4-treated group. Conclusion: Ex-4 does not affect biochemical, histopathological or genetic markers of pancreatitis in HFD induced mice. However, surveillance for a possible increased risk of pancreatitis under excess GLP-1RAs administration is warranted to ensure at least benefits far outweigh potential risks.

Exendin-4 attenuates neuronal death via GLP-1R/PI3K/Akt pathway in early brain injury after subarachnoid hemorrhage in rats

Neuronal apoptosis is considered to be a crucial therapeutic target against early brain injury (EBI) after subarachnoid hemorrhage (SAH). Emerging evidence indicates that Exendin-4 (Ex-4), a glucagon-like peptide 1 receptor (GLP-1R) agonist, plays a neuroprotective role in cerebrovascular disease. This study was conducted in order to verify the neuroprotective role of EX-4 in EBI after SAH in rats. The endovascular perforation model of SAH was performed in Sprague-Dawley rats (n = 153). Ex-4 was intraperitoneally injected 1 h after SAH induction in the rats (SAH + Ex-4). To elucidate the underlying molecular mechanism, small interfering ribonucleic acid (siRNA) for GLP-1R and a specific inhibitor of PI3K, LY294002, were injected intracerebroventricularly into SAH + Ex-4 rats before induction of SAH (n = 6 per group). SAH grading evaluation, immunohistochemistry, Western blots, neurobehavioral assessment, and Fluoro-Jade C (FJC) staining experiments were performed. Expression of GLP-1R was significantly increased and mainly expressed in neurons at 24 h after SAH induction. Administration of Ex-4 significantly improved both short- and long-term neurobehavior in SAH + Ex-4 group compared to SAH + Vehicle group after SAH. Ex-4 treatment significantly increased the expression of GLP-1R, PI3K, p-Akt, Bcl-xl, and Bcl-2, while at the same time was found to decrease expression of Bax in the brain. Effects of Ex-4 were reversed by the intervention of GLP-1R siRNA and LY294002 in SAH + Ex-4+GLP-1R siRNA and SAH + Ex-4+LY294002 groups, respectively. In conclusion, the neuroprotective effect of Ex-4 in EBI after SAH was mediated by attenuation of neuronal apoptosis via GLP-1R/PI3K/Akt signaling pathway, therefore EX-4 should be further investigated as a potential therapeutic agent in stroke patients.

Age-dependent human β cell proliferation induced by glucagon-like peptide 1 and calcineurin signaling.

Inadequate pancreatic β cell function underlies type 1 and type 2 diabetes mellitus. Strategies to expand functional cells have focused on discovering and controlling mechanisms that limit the proliferation of human β cells. Here, we developed an engraftment strategy to examine age-associated human islet cell replication competence and reveal mechanisms underlying age-dependent decline of β cell proliferation in human islets. We found that exendin-4 (Ex-4), an agonist of the glucagon-like peptide 1 receptor (GLP-1R), stimulates human β cell proliferation in juvenile but not adult islets. This age-dependent responsiveness does not reflect loss of GLP-1R signaling in adult islets, since Ex-4 treatment stimulated insulin secretion by both juvenile and adult human β cells. We show that the mitogenic effect of Ex-4 requires calcineurin/nuclear factor of activated T cells (NFAT) signaling. In juvenile islets, Ex-4 induced expression of calcineurin/NFAT signaling components as well as target genes for proliferation-promoting factors, including NFATC1, FOXM1, and CCNA1. By contrast, expression of these factors in adult islet β cells was not affected by Ex-4 exposure. These studies reveal age-dependent signaling mechanisms regulating human β cell proliferation, and identify elements that could be adapted for therapeutic expansion of human β cells.

The anti-diabetic drug exenatide, a glucagon-like peptide-1 receptor agonist, counteracts hepatocarcinogenesis through cAMP-PKA-EGFR-STAT3 axis.

Epidemiological studies have demonstrated a close association of type 2 diabetes and hepatocellular carcinoma (HCC). Exenatide (Ex-4), a potent diabetes drug targeting glucagon-like peptide-1 receptor (GLP-1R), is protective against non-alcoholic fatty liver disease (NAFLD). However, the Ex-4 function and GLP-1R status have yet been explored in HCC. Herein we investigated the effect of Ex-4 in diethylnitrosamine (DEN)-treated mice consuming control or high-fat high-carbohydrate diet. Administration of Ex-4 significantly improved obesity-induced hyperglycemia and hyperlipidemia and reduced HCC multiplicity in obese DEN-treated mice, in which suppressed proliferation and induced apoptosis were confined to tumor cells. The tumor suppression effects of Ex-4 were associated with high expression of GLP-1R and activation of cyclic AMP (cAMP) and protein kinase A (PKA). Importantly, Ex-4 also downregulated epidermal growth factor receptor (EGFR) and signal transducer and activator of transcription 3 (STAT3), which lie downstream of cAMP-PKA signaling, resulting in suppression of multiple STAT3-targeted genes including c-Myc, cyclin D1, survivin, Bcl-2 and Bcl-xl. The growth inhibitory effects of Ex-4 were consistent in GLP-1R-abundant hepatoma cell lines and xenograft mouse model, wherein both PKA and EGFR had obligatory roles in mediating Ex-4 functions. In addition, Ex-4 also effectively suppressed inflammatory and fibrotic phenotypes in mice fed with methionine-choline-deficient (MCD) diet and choline-deficient ethionine-supplemented (CDE) diet, respectively. In summary, Ex-4 elicits protective functions against NAFLD and obesity-associated HCC through cAMP-PKA-EGFR-STAT3 signaling, suggesting its administration as a novel approach to reduce HCC risk in diabetic patients.

Exendin-4 in combination with adipose-derived stem cells promotes angiogenesis and improves diabetic wound healing

BackgroundDiminished wound healing is a major complication of diabetes mellitus and can lead to foot ulcers. However, there are limited therapeutic methods to treat this condition. Exendin-4 (Ex-4), a glucagon-like peptide-1 receptor agonist, is known to have many beneficial effects on diabetes. In addition, mesenchymal stem cells are known to have wound healing effects. We investigated the effects of Ex-4 in combination with human adipose tissue-derived stem cells (ADSCs) on diabetic wound healing in a diabetic animal model.MethodsDiabetic db/db (blood glucose levels, >500 mg/dl) or C57BL/6 mice were subjected to wounding on the skin of the back. One day after wounding, each wound received ADSCs (2.5 × 105 cells) injected intradermally around the wound and/or Ex-4 (50 μl of 100 nM Ex-4) topically applied on the wound with a fine brush daily. Wound size was monitored and wound histology was examined. Human endothelial cells and keratinocyte cells were used to assess angiogenesis and vascular endothelial growth factor expression in vitro.ResultsTopical administration of Ex-4 or injection of ADSCs resulted in a rapid reduction of wound size in both diabetic and normoglycemic animals compared with vehicle treatment. Histological analysis also showed rapid skin reconstruction in Ex-4-treated or ADSC-injected wounds. A combination of Ex-4 and ADSCs showed a significantly better therapeutic effect over either treatment alone. In vitro angiogenesis assays showed that both Ex-4 and ADSC-conditioned media (CM) treatment improved migration, invasion and proliferation of human endothelial cells. ADSC-CM also increased migration and proliferation of human keratinocytes. In addition, both Ex-4 and ADSC-CM increased the expression of vascular endothelial growth factor. Co-culture with ADSCs increased migration and proliferation of these cells similar to that found after ADSC-CM treatment.ConclusionsWe suggest that Ex-4 itself is effective for the treatment of diabetic skin wounds, and a combination of topical treatment of Ex-4 and injection of ADSCs has a better therapeutic effect. Thus, a combination of Ex-4 and ADSCs might be an effective therapeutic option for the treatment of diabetic wounds, such as foot ulcers.

Suppression of ROS Production by Exendin-4 in PSC Attenuates the High Glucose-Induced Islet Fibrosis.

Pancreatic stellate cells (PSCs) play a major role to fibrotic islet destruction observed in diabetic patients and animal model of diabetes. Exendin-4 (Ex-4) is a potent insulinotropic agent and has been approved for the treatment of type 2 diabetes. However, there have been no reports demonstrating the effects of Ex-4 on pancreatic islet fibrosis. In this study, Ex-4 treatment clearly attenuated fibrotic islet destruction and improved glucose tolerance and islet survival. GLP-1 receptor expression was upregulated during activation and proliferation of PSCs by hyperglycemia. The activation of PKA pathway by Ex-4 plays a role in ROS production and angiotensin II (Ang II) production. Exposure to high glucose stimulated ERK activation and Ang II-TGF- β1 production in PSCs. Interestingly, Ex-4 significantly reduced Ang II and TGF-β1 production by inhibition of ROS production but not ERK phosphorylation. Ex-4 may be useful not only as an anti-diabetic agent but also as an anti-fibrotic agent in type 2 diabetes due to its ability to inhibit PSC activation and proliferation and improve islet fibrosis in OLETF rats.