Effects Of Ex-4 Research Articles

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.

Diabetes negatively affects cortical and striatal GABAergic neurons: an effect that is partially counteracted by exendin-4.

Type 2 diabetic (T2D) patients often develop early cognitive and sensorimotor impairments. The pathophysiological mechanisms behind these problems are largely unknown. Recent studies demonstrate that dysfunctional γ-aminobutyric acid (GABAergic) neurons are involved in age-related cognitive decline. We hypothesized that similar, but earlier dysfunction is taking place under T2D in the neocortex and striatum (two brain areas important for cognition and sensorimotor functions). We also hypothesized that the T2D-induced effects are pharmacologically reversible by anti-diabetic drugs targeting the glucagon-like peptide-1 receptor (GLP-1R). We determined the effect of T2D on cortical and striatal GABAergic neurons positive for glutamic acid decarboxylase-67 (GAD67), calbindin (CB), parvalbumin (PV) and calretinin (CR) by using immunohistochemistry and quantitative microscopy. Young and middle-aged T2D Goto-Kakizaki (GK) (a model of spontaneous T2D) and Wistar rats were used. Furthermore, we determined the therapeutic potential of the GLP1-R agonist exendin-4 (Ex-4) by treating middle-aged GK rats for 6weeks with 0.1μg/kg Ex-4 twice daily. We show that T2D reduced the density of GAD67-positive neurons in the striatum and of CB-positive neurons in both striatum and neocortex. T2D also increased the average volume of PV-positive interneurons in the striatum. Ex-4 treatment increased the density of CB-positive neurons in the striatum of GK rats. Our data demonstrate that T2D negatively affects GAD67 and CB-positive GABAergic neurons in the brain during aging, potentially identifying some of the pathophysiological mechanisms to explain the increased prevalence of neurological complications in T2D. We also show a specific, positive effect of Ex-4 on striatal CB-positive neurons, which could be exploited in therapeutic perspective.

The glucagon-like peptide-1 receptor agonist exendin-4 ameliorates warfarin-associated hemorrhagic transformation after cerebral ischemia.

BackgroundAs the number of patients with cardioembolic ischemic stroke is predicted to be double by 2030, increased burden of warfarin-associated hemorrhagic transformation (HT) after cerebral ischemia is an expected consequence. However, thus far, no effective treatment strategy is available for HT prevention in routine clinical practice. While the glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 (Ex-4) is known to protect against oxidative stress and neuronal cell death caused by ischemic brain damage, its effect on preventing warfarin-associated HT after cerebral ischemia is yet unknown. Therefore, we hypothesized that Ex-4 would stabilize the blood-brain barrier (BBB) and suppress neuroinflammation through PI3K-Akt-induced inhibition of glycogen synthase kinase-3β (GSK-3β) after warfarin-associated HT post-cerebral ischemia.MethodsWe used male C57BL/6 mice for all experiments. A 5-mg warfarin sodium tablet was dissolved in animals’ drinking water (effective warfarin uptake 0.04 mg (2 mg/kg) per mouse). The mice were fed for 0, 6, 12, and 24 h with ad libitum access to the treated water. To study the effects of Ex-4, temporary middle cerebral artery occlusion (MCAO) was performed. Then, either Ex-4 (10 mg/kg) or saline was injected through the tail vein, and in the Ex-4 + wortmannin group, PI3K inhibitor wortmannin was intravenously injected, after reperfusion. The infarct volume, neurological deficits, and integrity of the BBB were assessed 72 h post MCAO. One- or two-way ANOVA was used to test the difference between means followed by Newman–Keuls post hoc testing for pair-wise comparison.ResultsWe observed that Ex-4 ameliorated warfarin-associated HT and preserved the integrity of the BBB after cerebral ischemia through the PI3K/Akt/GSK-3β pathway. Furthermore, Ex-4 suppressed oxidative DNA damage and lipid peroxidation, attenuated pro-inflammatory cytokine expression levels, and suppressed microglial activation and neutrophil infiltration in warfarin-associated HT post-cerebral ischemia. However, these effects were totally abolished in the mice treated with Ex-4 + the PI3K inhibitor—wortmannin. The PI3K/Akt-GSK-3β signaling pathway appeared to contribute to the protection afforded by Ex-4 in the warfarin-associated HT model.ConclusionsGLP-1 administration could reduce warfarin-associated HT in mice. This beneficial effect of GLP-1 is associated with attenuating neuroinflammation and BBB disruption by inactivating GSK-3β through the PI3K/Akt pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0661-0) contains supplementary material, which is available to authorized users.

Effect of Exendin-4 on Autophagy Clearance in Beta Cell of Rats with Tacrolimus-induced Diabetes Mellitus.

Growing evidence suggests that GLP-1 protects beta cells against various cellular injuries by modulating autophagy. In this study, we examined whether exendin-4 (Ex-4), a GLP-1 analog, had preventive effects on tacrolimus (Tac)-induced beta cell injury by improving autophagy clearance. Rats with Tac-induced diabetes mellitus exhibited increased autophagy-associated protein expression, light chain 3B levels, and autophagic vacuole numbers in pancreatic beta cells. Additionally, Tac increased autophagy in a dose- and time-dependent manner in vitro, and inhibition of autophagosome using 3-methyladenine reduced Tac-induced islet cell injury by decreasing reactive oxygen species production and apoptosis. Ex-4 treatment decreased Tac-induced hyperglycaemia, oxidative stress, and apoptosis, accompanied by decreased autophagy-associated protein expression and autophagosome numbers. In vivo and in vitro studies showed that Tac treatment impaired lysosomal function and autophagosome-lysosome fusion; these processes were improve by Ex-4 treatment. Moreover, addition of bafilomycin A1, an inhibitor of lysosomal function, abolished the protective effects of Ex-4. Our findings reveal that Tac-induced diabetes mellitus was a state of excessive burden of autophagosomes and impairment of autophagy clearance and that Ex-4 protected against Tac-induced pancreatic islet injury by reducing the burden of autophagosomes via activation of autophagosome clearance. Thus, Ex-4 had therapeutic effects on Tac-induced pancreatic beta cell injury.

Unforeseen role for glucocorticoids in combinatorial anti-obesity pharmacology.

Unforeseen role for glucocorticoids in combinatorial anti-obesity pharmacology.

Role of lateral septum glucagon-like peptide 1 receptors in food intake.

Hindbrain glucagon-like peptide 1 (GLP-1) neurons project to numerous forebrain areas, including the lateral septum (LS). Using a fluorescently labeled GLP-1 receptor (GLP-1R) agonist, Exendin 4 (Ex4), we demonstrated GLP-1 receptor binding throughout the rat LS. We examined the feeding effects of Ex4 and the GLP-1R antagonist Exendin (9-39) (Ex9) at doses subthreshold for effect when delivered to the lateral ventricle. Intra-LS Ex4 suppressed overnight chow and high-fat diet (HFD) intake, and Ex9 increased chow and HFD intake relative to vehicle. During 2-h tests, intra-LS Ex9 significantly increased 0.25 M sucrose and 4% corn oil. Ex4 can cause nausea, but intra-LS administration of Ex4 did not induce pica. Furthermore, intra-LS Ex4 had no effect on anxiety-like behavior in the elevated plus maze. We investigated the role of LS GLP-1R in motivation for food by examining operant responding for sucrose on a progressive ratio (PR) schedule, with and without a nutrient preload to maximize GLP-1 neuron activation. The preload strongly suppressed PR responding, but blockade of GLP-1R in the intermediate subdivision of the LS did not affect motivation for sucrose under either load condition. The ability of the nutrient load to suppress subsequent chow intake was significantly attenuated by intermediate LS Ex9 treatment. By contrast, blockade of GLP-1R in the dorsal subdivision of the LS increased both PR responding and overnight chow intake. Together, these studies suggest that endogenous activity of GLP-1R in the LS influence feeding, and dLS GLP-1Rs, in particular, play a role in motivation.

Protective Effect of a GLP-1 Analog on Ischemia-Reperfusion Induced Blood-Retinal Barrier Breakdown and Inflammation.

PurposeInflammation associated with blood–retinal barrier (BRB) breakdown is a common feature of several retinal diseases. Therefore, the development of novel nonsteroidal anti-inflammatory approaches may provide important therapeutic options. Previous studies demonstrated that inhibition of dipeptidyl peptidase-IV, the enzyme responsible for the degradation of glucagon-like peptide-1 (GLP-1), led to insulin-independent prevention of diabetes-induced increases in BRB permeability, suggesting that incretin-based drugs may have beneficial pleiotropic effects in the retina. In the current study, the barrier protective and anti-inflammatory properties of exendin-4 (Ex-4), an analog of GLP-1, after ischemia-reperfusion (IR) injury were examined.MethodsIschemia-reperfusion injury was induced in rat retinas by increasing the intraocular pressure for 45 minutes followed by 48 hours of reperfusion. Rats were treated with Ex-4 prior to and following IR. Blood–retinal barrier permeability was assessed by Evans blue dye leakage. Retinal inflammatory gene expression and leukocytic infiltration were measured by qRT-PCR and immunofluorescence, respectively. A microglial cell line was used to determine the effects of Ex-4 on lipopolysaccharide (LPS)-induced inflammatory response.ResultsExendin-4 dramatically reduced the BRB permeability induced by IR injury, which was associated with suppression of inflammatory gene expression. Moreover, in vitro studies showed that Ex-4 also reduced the inflammatory response to LPS and inhibited NF-κB activation.ConclusionsThe present work suggests that Ex-4 can prevent IR injury–induced BRB breakdown and inflammation through inhibition of inflammatory cytokine production by activated microglia and may provide a novel option for therapeutic intervention in diseases involving retinal inflammation.

Limiting glucocorticoid secretion increases the anorexigenic property of Exendin-4.

ObjectiveGlucagon-like peptide-1 (GLP-1) analogs are attractive options for the treatment of type II diabetes and obesity because of their incretin and anorexigenic effects. Peripheral administration of the GLP-1R agonist Exendin-4 (Ex-4) also increases glucocorticoid secretion in rodents and humans, but whether the released glucocorticoids interact with Ex-4's anorexigenic effect remains unclear.MethodsTo test this, we used two experimental approaches that suppress corticosterone secretion and then assessed Ex-4 effects on eating in adult male rats. First, we combined acute and chronic low dose dexamethasone treatment with Ex-4. Second, we ablated hindbrain catecholamine neurons projecting to the hypothalamus with anti-dopamine-β-hydroxylase-saporin (DSAP) to block Ex-4-induced corticosterone secretion.ResultsCombining dexamethasone and Ex-4 produced a larger acute anorexigenic effect than Ex-4 alone. Likewise, chronic dexamethasone and Ex-4 co-treatment produced a synergistic effect on eating and greater body weight loss in diet-induced obese rats than Ex-4 alone. DSAP lesions not only blunted Ex-4's ability to increase corticosterone secretion, but potentiated the anorexigenic effect of Ex-4, indicating that Ex-4-dependent corticosterone secretion opposes Ex-4's actions. Consistent with the enhancement of Ex-4's anorexigenic effect, DSAP lesion altered Ex-4-dependent changes in neuropeptide Y, preproglucagon, and corticotropin releasing hormone gene expression involved in glucocorticoid feedback.ConclusionsOur findings demonstrate that limiting glucocorticoid secretion and actions with low dose dexamethasone or DSAP lesion increases Ex-4's ability to reduce food intake and body weight. Novel glucocorticoid receptor based mechanisms, therefore, may help enhance GLP-1-based obesity therapies.

Exendin-4 promotes proliferation of adipose-derived stem cells through ERK and JNK signaling pathways.

Adipose-derived stem cell (ADSC) transplantation has emerged as a potential tool for the treatment of cardiovascular disease. However, with a limited renewal capacity and the need for mass cells during the engraftment, strategies are needed to enhance ADSC proliferative capacity. In this study, we explored the effects of exendin-4 (Ex-4), a glucagon-like peptide-1 analog, on the growth of ADSCs, focusing in particular on c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) signaling pathways. Firstly, ADSCs were isolated and cultured in vitro. Then, flow cytometry demonstrated that ADSCs were positive for CD90 and CD29 but negative for CD31, CD34, and CD45. Ex-4 (0-50nM) treatment increased ADSC proliferation in a dose-dependent manner but had no effects on stem cell markers of ADSCs. Moreover, we found that Ex-4 treatment elevated the phosphorylation levels of the JNK and ERK signaling pathways. Furthermore, utilization of Ex-4 also promoted cyclin D1 and cyclin E protein expression, which was accompanied by more Edu(+) cells and a higher percentage of cells in the S-phase of the cell cycle after Ex-4 treatment. In parallel, the application of inhibitors SP600125 and PD98059, inhibitors of the JNK and ERK signaling pathways, respectively, not only reversed such effects of Ex-4 on JNK and ERK but also resulted in lower percentages of S-phase cells and fewer numbers of Edu(+) cells. In summary, Ex-4 has no effects on stem cell markers in ADSCs but promotes ADSC growth via JNK and ERK signaling pathways.

Exendin-4 protects bone marrow-derived mesenchymal stem cells against oxygen/glucose and serum deprivation-induced apoptosis through the activation of the cAMP/PKA signaling pathway and the attenuation of ER stress

Exendin-4 (ex-4) is a long-acting glucagon-like peptide-1 receptor (GLP-1R) agonist which exerts beneficial effects on glycemic control and promotes cell viability. In the present study, we investigated the anti-apoptotic effects of ex-4, as well as the potential mechanisms responsible for these effects in rat bone marrow-derived mesenchymal stem cells (BM-MSCs) under conditions of oxygen, glucose and serum deprivation (OGD). The apoptosis of the MSCs was induced by subjecting the cells to OGD conditions for 4 h and was detected by Annexin V/PI and Hoechst 33258 staining. The MSCs were pre-conditioned with ex-4 for 12 h prior to being subjected to OGD conditions, and the expression levels of an apoptotic marker (cleaved caspase-3), endoplasmic reticulum (ER) stress markers [phosphorylated (p-)protein kinase RNA-like endoplasmic reticulum kinase (PERK), PERK, binding immunoglobulin protein (BIP), activating transcription factor 4 (ATF-4) and C/EBP homologous protein (CHOP)], as well as those of a survival marker (Bcl-2) were measured by western blot analysis. Furthermore, the mRNA levels of ATF-4 and CHOP were determined by RT-qPCR. ELISA was used to examine the activity of intracellular cAMP. Moreover, the GLP-1R antagonist, exendin9-39 (ex9-39), the protein kinase A (PKA) inhibitor, H89, and small interfering RNA (siRNA) targeting rat ATF-4 and CHOP were co-incubated with the MSCs. The apoptotic rate was markedly diminished following pre-conditioning with ex-4 in a dose-dependent manner (P<0.05). The ER stress markers, p-PERK, BIP, ATF-4 and CHOP, were upregulated in the cells subjected to OGD conditions. Ex-4 pre-conditioning significantly decreased the mRNA and protein levels of ATF-4 and CHOP (P<0.05), and increased the activity of intracellular cAMP (P<0.05). Furthermore, the anti-apoptotic effects of ex-4 were almost reversed by treatment with either H89 or ex9-39 (P<0.05); transfection with siRNA-CHOP significantly reduced the apoptotic rate of the MSCs and did not impair the cytoprotective effects of ex-4. Taken together, these findings suggest that ex-4 protects rat BM-MSCs from OGD-induced apoptosis through the activation of the PKA/cAMP pathway and the attenuation of the ER stress signaling pathway. Ex-4 may thus prove to be a therapeutic agent with the potential to improve the viability of MSCs in the ischemic milieu, and consequently, to optimize the therapeutic effects of MSC therapy in acute myocardial infarction.

Sex and estrogens alter the action of glucagon-like peptide-1 on reward.

BackgroundFeeding behavior is regulated through an intricate array of anorexic and orexigenic hormones acting on the central nervous system (CNS). Some of these hormones may have differential effects in males and females, effects potentially attributed to actions of gonadal steroids, especially estrogens. Central stimulation of the glucagon-like peptide-1 (GLP-1) receptors reduces feeding and food-reward behavior by acting on CNS regions important for the anorexic actions of estrogens. Thus, we propose that the action of GLP-1 on food intake and reward may differ between sexes.MethodsMale and female rats were centrally injected with the GLP-1 analog exendin-4 (Ex4) in a non-deprived or food-restricted state; reward behavior was measured in a progressive ratio operant conditioning task. Intake of chow and palatable food were also measured. To determine if sex differences in the actions of Ex4 are due to interactions with estrogens, Ex4 treatment was preceded by treatment with a nonselective estrogen receptor-α (ERα) and ERβ or ERα-selective antagonist.ResultsCentral injection of Ex4 revealed increased reward behavior suppression in females, compared to males, in the operant conditioning task. This increase was present in both non-deprived and food-restricted animals with larger differences in the fed state. Intake of chow and palatable food, after Ex4, were similar in males and females. Food reward, but not food intake, effect of Ex4 was attenuated by pretreatment with ER antagonist in both sexes, suggesting that estrogens may modulate effects of Ex4 in both sexes. Furthermore, central pretreatment with ERα-selective antagonist was sufficient to attenuate effects of Ex4 on reward.ConclusionsCollectively, these data reveal that females display much higher sensitivity to the food reward impact of central GLP-1 receptor activation. Surprisingly, they also demonstrate that central ERα signaling is necessary for the actions of GLP-1 on food-reward behavior in both sexes.

Differential effects of GLP-1 receptor agonist on foam cell formation in monocytes between non-obese and obese subjects

ObjectiveMonocytes/macrophages (Mϕ) transform into foam cells in the presence of oxidized-LDL (ox-LDL), releasing inflammatory mediators. The antiatherogenic role of a dipeptidyl peptidase-4 inhibitor is mediated, in part, through improving the unbalance of inflammatory (M1)/anti-inflammatory (M2) phenotypes in monocytes. In this study, we examined differential regulation of glucagon-like peptide-1 receptor (GLP-1R) signaling for antiatherogenesis in monocytes/Mϕ from normal-weight control subjects and obese patients. MethodsWe evaluated the effects of exendin-4 (Ex-4), a GLP-1R agonist, on ox-LDL-stimulated foam cell formation, M1/M2 cytokine production, and organelle change in primary monocytes from control subjects and obese patients and human monocytic THP-1-derived Mϕ as well. ResultsHere we report that Ex-4 suppressed foam cell formation and M1 cytokine expression and, interestingly, induced indicators of autophagy in ox-LDL-stimulated monocytes from control subjects. The suppressing effects on foam cell formation by Ex-4 were reversed by a cAMP inhibitor. In contrast to control subjects, Ex-4 did not induce indicators of autophagy, but did induce foam cell formation and M1 cytokine expression in monocytes from obese patients. GLP-1R expression level was comparable between control subjects and obese patients. The effects of Ex-4 on inducing indicators of autophagy and suppressing foam cell formation were observed in THP-1 Mϕ. ConclusionsThese data suggest that GLP-1R signaling induces autophagy, thereby suppressing foam cell formation in non-obese subjects. In obese patients, GLP-1R stimulation increased foam cell formation and IL-6, TNF-α, and IL-1β production. Such altered signaling in monocytes of obese patients may be involved in the development of atherosclerosis.

Short term exendin-4 treatment reduces markers of metabolic disorders in female offspring of obese rat dams

ObjectivesMaternal obesity imposes significant health risks in the offspring including diabetes and dyslipidemia. We previously showed that the hypoglycaemic agent exendin-4 (Ex-4) administered from weaning can reverse the maternal impact of ‘transmitted disorders’ in such offspring. However daily injection for six-weeks was required and the beneficial effect may lapse upon drug withdrawal. This study aimed to investigate whether short term Ex-4 treatment during suckling period in a rodent model can reverse transmitted metabolic disorders due to maternal obesity. MethodsMaternal obesity was induced in female Sprague Dawley rats by high-fat diet feeding for 6 weeks, throughout gestation and lactation. Female offspring were treated with Ex-4 (5μg/kg/day) between postnatal day (P) 4 and 14. Female offspring were harvested at weaning (P20). Lipid and glucose metabolic markers were measured in the liver and fat. Appetite regulators were measured in the plasma and hypothalamus. ResultsMaternal obesity significantly increased body weight, fat mass, and liver weight in the offspring. There was an associated inhibition of peroxisomal proliferator activated receptor gamma coactivator 1α (PGC1α), increased fatty acid synthase (FASN) expression in the liver, and reduced adipocyte triglyceride lipase (ATGL) expression. It also increased the plasma gut hormone ghrelin and reduced glucagon-like peptide-1. Ex-4 treatment partially reversed the maternal impact on adiposity and impaired lipid metabolism in the offspring, with increased liver PGC1α and inhibition of FASN mRNA expression. Ex-4 treatment also increased the expression of a novel fat depletion gene a2-zinc-glycoprotein 1 in the fat tissue. ConclusionShort term Ex-4 treatment during the suckling period significantly improved the metabolic profile in the offspring from the obese mothers at weaning. Long-term studies are needed to follow such offspring to adulthood to examine the sustained effects of Ex-4 in preventing the development of metabolic disease.

Effects of Exendin-4 on bone marrow mesenchymal stem cell proliferation, migration and apoptosis in vitro.

Mesenchymal stem cells (MSC) are regarded as an attractive source of therapeutic stem cells for myocardial infarction. However, their limited self-renewal capacity, low migration capacity and poor viability after transplantation hamper the clinical use of MSC; thus, a strategy to enhance the biological functions of MSC is required. Exendin-4 (Ex-4), a glucagon-like peptide-1 receptor agonist, exerts cell-protective effects on many types of cells. However, little information is available regarding the influence of Ex-4 on MSC. In our study, MSC were isolated from bone marrow and cultured in vitro. After treatment with Ex-4, MSC displayed a higher proliferative capacity, increased C-X-C motif receptor 4 (CXCR4) expression and an enhanced migration response. Moreover, in H2O2-induced apoptosis, Ex-4 preserved mitochondrial function through scavenging ROS and balancing the expression of anti- and pro-apoptotic proteins, leading to the inhibition of the mitochondria-dependent cell death pathways and increased cell survival. Moreover, higher phospho-Akt (p-Akt) expression was observed after Ex-4 intervention. However, blockade of the PI3K/Akt pathway with inhibitors suppressed the above cytoprotective effects of Ex-4, suggesting that the PI3K/Akt pathway is partly responsible for Ex-4-mediated MSC growth, mobilization and survival. These findings provide an attractive method of maximizing the effectiveness of MSC-based therapies in clinical applications.

Exendin-4 Enhances Motor Function Recovery via Promotion of Autophagy and Inhibition of Neuronal Apoptosis After Spinal Cord Injury in Rats.

Autophagy occurs prior to apoptosis and plays an important role in cell death regulation during spinal cord injury (SCI). This study aimed to determine the effects and potential mechanism of the glucagon-like peptide-1 (GLP-1) agonist extendin-4 (Ex-4) in SCI. Seventy-two male Sprague Dawley rats were randomly assigned to sham, SCI, 2.5μg Ex-4, and 10μg Ex-4 groups. To induce SCI, a 10-g iron rod was dropped from a 20-mm height to the spinal cord surface. Ex-4 was administered via intraperitoneal injection immediately after surgery. Motor function evaluation with the Basso Beattie Bresnahan (BBB) locomotor rating scale indicated significantly increased scores (p < 0.01) in the Ex-4-treated groups, especially 10μg, which demonstrated the neuroprotective effect of Ex-4 after SCI. The light chain 3-II (LC3-II) and Beclin 1 protein expression determined via western blot and the number of autophagy-positive neurons via immunofluorescence double labeling were increased by Ex-4, which supports promotion of autophagy (p < 0.01). The caspase-3 protein level and neuronal apoptosis via transferase UTP nick end labeling (TUNEL)/NeuN/DAPI double labeling were significantly reduced in the Ex-4-treated groups, which indicates anti-apoptotic effects (p < 0.01). Finally, histological assessment via Nissl staining demonstrated the Ex-4 groups exhibited a significantly greater number of surviving neurons and less cavity (p < 0.01). To our knowledge, this is the first study to indicate that Ex-4 significantly enhances motor function in rats after SCI, and these effects are associated with the promotion of autophagy and inhibition of apoptosis.

Parabrachial Nucleus Contributions to Glucagon-Like Peptide-1 Receptor Agonist-Induced Hypophagia.

Exendin-4 (Ex4), a glucagon-like peptide-1 receptor (GLP-1R) agonist approved to treat type 2 diabetes mellitus, is well known to induce hypophagia in human and animal models. We evaluated the contributions of the hindbrain parabrachial nucleus (PBN) to systemic Ex4-induced hypophagia, as the PBN receives gustatory and visceral afferent relays and descending input from several brain nuclei associated with feeding. Rats with ibotenic-acid lesions targeted to the lateral PBN (PBNx) and sham controls received Ex4 (1 μg/kg) before 24 h home cage chow or 90 min 0.3 M sucrose access tests, and licking microstructure was analyzed to identify components of feeding behavior affected by Ex4. PBN lesion efficacy was confirmed using conditioned taste aversion (CTA) tests. As expected, sham control but not PBNx rats developed a CTA. In sham-lesioned rats, Ex4 reduced chow intake within 4 h of injection and sucrose intake within 90 min. PBNx rats did not show reduced chow or sucrose intake after Ex4 treatment, indicating that the PBN is necessary for Ex4 effects under the conditions tested. In sham-treated rats, Ex4 affected licking microstructure measures associated with hedonic taste evaluation, appetitive behavior, oromotor coordination, and inhibitory postingestive feedback. Licking microstructure responses in PBNx rats after Ex4 treatment were similar to sham-treated rats with the exception of inhibitory postingestive feedback measures. Together, the results suggest that the PBN critically contributes to the hypophagic effects of systemically delivered GLP-1R agonists by enhancing visceral feedback.

Exendin-4 protects adipose-derived mesenchymal stem cells from apoptosis induced by hydrogen peroxide through the PI3K/Akt–Sfrp2 pathways

Adipose-derived mesenchymal stem cells (ADMSCs)-based therapy is a promising modality for the treatment of myocardial infarction in the future. However, the majority of transplanted cells are readily lost after transplantation because of hypoxia and oxidative stress. An efficient means to enhance the ability of ADMSCs to survive under pathologic conditions is required. In our study, we explored the effects of exendin-4 (Ex-4) on ADMSCs apoptosis in vitro induced by hydrogen peroxide, focusing in particular on mitochondrial apoptotic pathways and PI3K/Akt–secreted frizzled-related protein 2 (Sfrp2) survival signaling. We demonstrated that ADMSCs subjected to H2O2 for 12h exhibited impaired mitochondrial function and higher apoptotic rate. However, Ex-4 (1–20nM) preconditioning for 12h could protect ADMSCs against H2O2-mediated apoptosis in a dose-dependent manner. Furthermore, Ex-4 pretreatment upregulated the levels of superoxide dismutase and glutathione as well as downregulating the production of reactive oxygen species and malondialdehyde. Western blots revealed that increased antiapoptotic proteins Bcl-2 and c-IAP1/2 as well as decreased proapoptotic proteins Bax and cytochrome c appeared in ADMSCs with Ex-4 incubation, which inhibited the caspase-9-involved mitochondrial apoptosis pathways with evidence showing inactivation of caspase-9/3 and preservation of mitochondrial membrane potential. Furthermore, we illustrated that Ex-4 enhanced Akt phosphorylation, which increased the expression of Sfrp2. Notably, blockade of the PI3K/Akt pathway or knockdown of Sfrp2 with siRNA obviously abolished the protective effects of Ex-4 on mitochondrial function and ADMSCs apoptosis under H2O2. In summary, this study confirmed that H2O2 induced ADMSCs apoptosis through mitochondria-dependent cell death pathways, and Ex-4 preconditioning may reduce such apoptosis of ADMSCs through the PI3K/Akt–Sfrp2 pathways.

Exendin-4, a GLP-1 receptor agonist, attenuates prostate cancer growth.

Recently, pleiotropic benefits of incretin therapy beyond glycemic control have been reported. Although cancer is one of the main causes of death in diabetic patients, few reports describe the anticancer effects of incretin. Here, we examined the effect of the incretin drug exendin (Ex)-4, a GLP-1 receptor (GLP-1R) agonist, on prostate cancer. In human prostate cancer tissue obtained from patients after they had undergone radical prostatectomy, GLP-1R expression colocalized with P504S, a marker of prostate cancer. In in vitro experiments, Ex-4 significantly decreased the proliferation of the prostate cancer cell lines LNCap, PC3, and DU145, but not that of ALVA-41. This antiproliferative effect depended on GLP-1R expression. In accordance with the abundant expression of GLP-1R in LNCap cells, a GLP-1R antagonist or GLP-1R knockdown with small interfering RNA abolished the inhibitory effect of Ex-4 on cell proliferation. Although Ex-4 had no effect on either androgen receptor activation or apoptosis, it decreased extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase (MAPK) phosphorylation in LNCap cells. Importantly, Ex-4 attenuated in vivo prostate cancer growth induced by transplantation of LNCap cells into athymic mice and significantly reduced the tumor expression of P504S, Ki67, and phosphorylated ERK-MAPK. These data suggest that Ex-4 attenuates prostate cancer growth through the inhibition of ERK-MAPK activation.

Mitigation of autophagy ameliorates hepatocellular damage following ischemia-reperfusion injury in murine steatotic liver.

Ischemia-reperfusion injury (IRI) is a common clinical consequence of hepatic surgery, cardiogenic shock, and liver transplantation. A steatotic liver is particularly vulnerable to IRI, responding with extensive hepatocellular injury. Autophagy, a lysosomal pathway balancing cell survival and cell death, is engaged in IRI, although its role in IRI of a steatotic liver is unclear. The role of autophagy was investigated in high-fat diet (HFD)-fed mice exposed to IRI in vivo and in steatotic hepatocytes exposed to hypoxic IRI (HIRI) in vitro. Two inhibitors of autophagy, 3-methyladenine and bafilomycin A1, protected the steatotic hepatocytes from HIRI. Exendin 4 (Ex4), a glucagon-like peptide 1 analog, also led to suppression of autophagy, as evidenced by decreased autophagy-associated proteins [microtubule-associated protein 1A/1B-light chain 3 (LC3) II, p62, high-mobility group protein B1, beclin-1, and autophagy-related protein 7], reduced hepatocellular damage, and improved mitochondrial structure and function in HFD-fed mice exposed to IRI. Decreased autophagy was further demonstrated by reversal of a punctate pattern of LC3 and decreased autophagic flux after IRI in HFD-fed mice. Under the same conditions, the effects of Ex4 were reversed by the competitive antagonist exendin 9-39. The present study suggests that, in IRI of hepatic steatosis, treatment of hepatocytes with Ex4 mitigates autophagy, ameliorates hepatocellular injury, and preserves mitochondrial integrity. These data suggest that therapies targeting autophagy, by Ex4 treatment in particular, may ameliorate the effects of IRI in highly prevalent steatotic liver.

Corticotropin-releasing hormone and the sympathoadrenal system are major mediators in the effects of peripherally administered exendin-4 on the hypothalamic-pituitary-adrenal axis of male rats.

Glucagon-like peptide-1 (GLP-1) and the GLP-1 receptor agonist, exendin-4 (Ex-4), potently stimulate hypothalamic-pituitary-adrenal (HPA) axis activity after either central or peripheral administration. Because several GLP-1 derivative drugs, including synthetic Ex-4, are currently in use to treat patients with type II diabetes mellitus, the characterization of Ex-4 effects on the HPA axis is highly relevant. Herein, the roles of CRH and AVP on these effects were investigated by administering the antagonists astressin and d(CH2)5Tyr(Me)AVP, respectively. The role of the sympathoadrenal system (SAS) was explored in bilateral adrenal enucleated and guanethidine-treated rats, whereas primary pituitary cell cultures were used to study direct effects on the corticotropes. Astressin completely abrogated (P < .05) the effects of Ex-4 central administration on ACTH secretion but only slightly reduced (by 35%) the ACTH response to Ex-4 peripheral administration. Moreover, astressin significantly (P < .05) decreased the corticosterone response to centrally but not peripherally administered Ex-4, suggesting different mechanisms depending on the route of administration. Pretreatment with d(CH2)5Tyr(Me)AVP failed to diminish either the ACTH or corticosterone response to Ex-4 and no direct effect of Ex-4 or GLP-1 was observed on pituitary cell cultures. In contrast, a significant (P < .05) reduction in the corticosterone response elicited by Ex-4 peripheral administration was observed in enucleated and guanethidine-treated rats, indicating a role of the SAS in the glucocorticoid stimulatory effects of Ex-4. Our data demonstrate that the effects of Ex-4 on the HPA axis are partially mediated by CRH and the sympathoadrenal system, and stress the relevance of Ex-4 as a corticosterone secretagogue.