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We previously found that coadministering peptides and proteins with the cell-penetrating peptide L-penetratin intranasally significantly increased transport to the brain and enhanced pharmacological effects. The present study aimed to clarify the mechanisms of nose-to-brain drug delivery enhancement by L-penetratin coadministration. First, we compared the concentrations of Exendin-4 in plasma and brain after intranasal and subcutaneous administration and suggested that coadministration with L-penetratin facilitated the direct nose-to-brain transport of Exendin-4. Second, we demonstrated that L-penetratin did not stimulate the transport of Cy7-labeled Exendin-4 and insulin through the trigeminal nerves but shifted their distribution to the olfactory mucosal pathway. Third, we investigated the distribution of insulin into the deeper regions of the brain after delivery via the olfactory pathway and suggested that insulin had entered the olfactory bulb, bottom part of the brain, and perivascular space through the cerebrospinal fluid and had diffused throughout the brain. We further demonstrated that intranasally delivered insulin with L-penetratin specifically accumulated on the hippocampus neuronal cells. Thus, this study suggested that administrating peptide drugs intranasally with L-penetratin allows direct transport to the olfactory bulb, bottom part of the brain, and perivascular space of the cerebral artery. This technique also potentially allows targeting of specific brain areas.

Exendin-4 (Ex-4) is a glucagon-like peptide-1 receptor (GLP-1R) agonist that protects against brain injury. However, little is known about the effect of Ex-4 on kainic acid (KA)-induced seizures and hippocampal cell death. Therefore, this study evaluated the neuroprotective effects of Ex-4 pretreatment in a mouse model of KA-induced seizures. Three days before KA treatment, mice were intraperitoneally injected with Ex-4. We found that Ex-4 pretreatment reversed KA-induced reduction of GLP-1R expression in the hippocampus and attenuated KA-induced seizure score, hippocampal neuronal death, and neuroinflammation. Ex-4 pretreatment also dramatically reduced hippocampal lipocalin-2 protein in KA-treated mice. Furthermore, immunohistochemical studies showed that Ex-4 pretreatment significantly alleviated blood–brain barrier leakage. Finally, Ex-4 pretreatment stimulated hippocampal expression of phosphorylated cyclic adenosine monophosphate (cAMP) response element-binding protein (p-CREB), a known target of GLP-1/GLP-1R signaling. These findings indicate that Ex-4 pretreatment may protect against KA-induced neuronal damage by regulating GLP-1R/CREB-mediated signaling pathways.

Regulated exocytosis establishes a narrow fusion pore as initial aqueous connection to the extracellular space, through which small transmitter molecules such as ATP can exit. Co-release of polypeptides and hormones like insulin requires further expansion of the pore. There is evidence that pore expansion is regulated and can fail in diabetes and neurodegenerative disease. Here, we report that the cAMP-sensor Epac2 (Rap-GEF4) controls fusion pore behavior by acutely recruiting two pore-restricting proteins, amisyn and dynamin-1, to the exocytosis site in insulin-secreting beta-cells. cAMP elevation restricts and slows fusion pore expansion and peptide release, but not when Epac2 is inactivated pharmacologically or in Epac2-/- (Rapgef4-/-) mice. Consistently, overexpression of Epac2 impedes pore expansion. Widely used antidiabetic drugs (GLP-1 receptor agonists and sulfonylureas) activate this pathway and thereby paradoxically restrict hormone release. We conclude that Epac2/cAMP controls fusion pore expansion and thus the balance of hormone and transmitter release during insulin granule exocytosis.

Tight control of beta cell stimulus-secretion coupling is crucial for maintaining homeostasis of energy-rich nutrients. While glucose serves as a primary regulator of this process, incretins augment beta cell function, partly by enhancing cytosolic [Ca 2+ ] dynamics. However, the details of how precisely they affect beta cell recruitment during activation, their active time, and functional connectivity during plateau activity, and how they influence beta cell deactivation remain to be described. Performing functional multicellular Ca 2+ imaging in acute mouse pancreas tissue slices enabled us to systematically assess the effects of the GLP-1 receptor agonist exendin-4 (Ex-4) simultaneously in many coupled beta cells with high resolution. In otherwise substimulatory glucose, Ex-4 was able to recruit approximately a quarter of beta cells into an active state. Costimulation with Ex-4 and stimulatory glucose shortened the activation delays and accelerated beta cell activation dynamics. More specifically, active time increased faster, and the time required to reach half-maximal activation was effectively halved in the presence of Ex-4. Moreover, the active time and regularity of [Ca 2+ ] IC oscillations increased, especially during the first part of beta cell response. In contrast, subsequent addition of Ex-4 to already active cells did not significantly enhance beta cell activity. Network analyses further confirmed increased connectivity during activation and activity in the presence of Ex-4, with hub cell roles remaining rather stable in both control experiments and experiments with Ex-4. Interestingly, Ex-4 demonstrated a biphasic effect on deactivation, slightly prolonging beta cell activity at physiological concentrations and shortening deactivation delays at supraphysiological concentrations. In sum, costimulation by Ex-4 and glucose increases [Ca 2+ ] IC during beta cell activation and activity, indicating that the effect of incretins may, to an important extent, be explained by enhanced [Ca 2+ ] IC signals. During deactivation, previous incretin stimulation does not critically prolong cellular activity, which corroborates their low risk of hypoglycemia., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Paradiž Leitgeb, Kerčmar, Križančić Bombek, Pohorec, Skelin Klemen, Slak Rupnik, Gosak, Dolenšek and Stožer.)

Background: Exendin-4 (Ex4) is a promising drug for diabetes mellitus with a half-life of 2.4 h in human bodies. Besides, the Ex4 formulations currently employed in the clinic or under development have problems pertaining to stability. In this study, palmitic acid-modified Ex4 (Pal-Ex4) was prepared and purified to extend the half-life of Ex4. In addition, Pal-Ex4-MVLs were further designed and optimized as a long-acting delivery system for intramuscular injection., Methods: Pal-Ex4 was encapsulated within multivesicular liposomes (MVLs) via a two-step double emulsification process. The formulated products were then assessed for their vesicle size, encapsulation efficiency, and in vitro and in vivo ., Results: Pal-Ex4-MVLs with a notable encapsulation efficiency of 99.18% were successfully prepared. Pal-Ex4-MVLs, administered via a single intramuscular injection in Sprague-Dawley rats, sustained stable plasma concentrations for 168 h, presenting extended half-life (77.28 ± 12.919 h) and enhanced relative bioavailability (664.18%). MVLs protected Ex4 through providing stable retention and slow release. This approach considerably improved the in-situ stability of the drug for intramuscular administration., Conclusions: The combination of palmitic acid modification process with MVLs provides dual protection for Ex4 and can be a promising strategy for other hydrophilic protein/polypeptide-loaded sustained-release delivery systems with high drug bioactivity.

Nonunions and delayed unions pose significant challenges in orthopedic treatment, with current therapies often proving inadequate. Bone tissue engineering (BTE), particularly through endochondral ossification (ECO), emerges as a promising strategy for addressing critical bone defects. This study introduces mesenchymal stem cells overexpressing Exendin-4 (MSC-E4), designed to modulate bone remodeling via their autocrine and paracrine functions. We established a type I collagen (Col-I) sponge-based in vitro model that effectively recapitulates the ECO pathway. MSC-E4 demonstrated superior chondrogenic and hypertrophic differentiation and enhanced the ECO cell fate in single-cell sequencing analysis. Furthermore, MSC-E4 encapsulated in microscaffold, effectively facilitated bone regeneration in a rat calvarial defect model, underscoring its potential as a therapeutic agent for bone regeneration. Our findings advocate for MSC-E4 within a BTE framework as a novel and potent approach for treating significant bone defects, leveraging the intrinsic ECO process., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Jianhao Lin reports financial support was provided by 10.13039/501100012401Beijing Science and Technology Planning Project. Dan Xing reports financial support was provided by 10.13039/501100004826Beijing Natural Science Foundation. Dan Xing reports financial support was provided by 10.13039/501100001809National Natural Science Foundation of China., (© 2023 The Authors.)

Background: Neuroprotective mechanisms triggered by peroxisome proliferator-activated receptor-gamma agonist: pioglitazone (PIO) and glucagon-like peptide 1 analog: exendin-4 (Ex-4) in neurological diseases were reported, but whether mitochondrial biogenesis is involved or not in their neuro-protective mechanisms in type 1 Diabetes Mellitus (T1DM); has not been studied before. To bridge this gap, we investigated the effect of PIO and Ex-4 on brain mitochondrial biogenesis in streptozotocin-induced diabetes in rats. Methods: Seven weeks after induction of diabetes in rats, serum fasting glucose and insulin were measured in studied groups. The brain was removed for histological analysis and assessment of: mitochondrial complexes I and II, ATP, H2O2, brain derived neurotrophic factor (BDNF), cytochrome c and hemeoxygenase (HO)-1 activity, and relative gene expression of the nuclear factor; Nrf2 and the apoptotic markers: bax & bcl2 and mitochondrial biogenesis markers; peroxisome proliferator-activated receptor γ coactivator (PGC) 1-a and sirtuin 1 (SIRT-1) and AMP-activated protein kinase (AMPK) and c-Jun-N-terminal kinase (JNK) proteins. Results: Brain in untreated rats showed neurodegeneration area and significantly rising H2O2 and JNK, upregulation of bax, down-regulation of bcl2. These changes were paralleled with significant reduction in Nrf2, HO-1, BDNF, complex I, II and ATP and SIRT-1/PGC1-a expression. PIO and Ex-4 significantly improved the reported changes. Combined modality showed better improvement relative to each drug alone. Conclusions: PIO and Ex-4 may have neuroprotective effects in T1DM, via targeting altered mitochondrial biogenesis probably due to modulation of brain SIRT-1 signaling, improvement of oxidative stress and equilibrating the balance between pro-apoptotic and anti-apoptotic mediators. [ABSTRACT FROM AUTHOR]

Glucagon-like peptide-1 (GLP-1) and its receptor (GLP-1R) exert cardioprotective effects after myocardial ischemia and reperfusion (MI/R) in animal models and human clinical trials. Receptor imaging with positron emission tomography (PET) provides a non-invasive method for monitoring GLP-1R expression. In the present study, a fluorine-18-labeled aluminum fluoride exendin-4 analog [18F-AlF conjugated with 1,4,7-triazacyclononanetriacetic acid (NOTA)-maleimide (MAL)-Cys40-exendin-4] was synthesized and evaluated in a rat MI/R model for GLP-1R imaging. NOTA-MAL-Cys40-exendin-4 was synthesized by coupling Cys40-exendin-4 with NOTA-MAL. NOTA-MAL-Cys40-exendin-4 was then conjugated with 18F-AlF to obtain 18F-AlF-NOTA-MAL-Cys40-exendin-4. The yield of 18F-AlF-NOTA-MAL-Cys40-exendin-4 was 18.5±3.4% (not decay corrected). The process was completed within ~30 min. In rat MI/R models, the tracer exhibited specific binding to GLP-1R and an appropriate signal-to-noise ratio. At 8 h post-MI/R, tracer uptake reached its peak [0.35±0.053% of injected dose (%ID)/g; n=6] in ischemic myocardium. Localized tracer uptake decreased 1 day (0.20±0.032 %ID/g; n=6) and 3 days (0.16±0.017 %ID/g; n=6) post-MI/R compared with 8 h post-MI/R, but still remained higher compared with sham-operated groups (0.06±0.012 %ID/g; n=6). Pre-injected unlabeled exendin-4 effectively blocked tracer accumulation (0.09±0.041 %ID/g; n=6). In conclusion, 18F-AlF-NOTA-MAL-Cys40-exendin-4 demonstrated favorable characteristics for GLP-1R imaging following MI/R. PET imaging using 18F-AlF-NOTA-MAL-Cys40-exendin-4 in rodent hearts after MI/R revealed a dynamic pattern of GLP-1R upregulation. [ABSTRACT FROM AUTHOR]

A growing body of evidence indicates that treatment with glucagon-like peptide-1 (GLP-1) receptor agonists can be beneficial for patients with osteoporosis. However, the underlying mechanism by which GLP-1 receptor agonists improve osteoporosis remains unclear. In this study, we assessed the anti-osteoporosis effects of Exendin-4, a highly potent GLP-1 receptor agonist, using a rat senescent osteoblast model. We found that Exendin-4 improved proliferation of senescent osteoblasts, as indicated by MTT assay and ALP activity detection. RT-qPCR revealed that Exendin-4 up-regulated the expression of bone metabolism genes (OPG, RANKL, BGP) and down-regulated the expression of aging-related genes (p16, p21, p53). Meanwhile, we observed a sustained increase in IGF-1 receptor (IGF-1R) expression, and not GLP-1 receptor (GLP-1R) expression, in senescent osteoblasts treated with Exendin-4. Furthermore, intervention with Exendin-4 in senescent osteoblasts increased IGF-1, p-PI3K, and p-Akt protein levels, as shown by western blot analysis. Finally, downregulation of IGF-1 by RNAi inhibited the anti-osteoporosis effects of Exendin-4, which is associated with the IGF-1/PI3K/Akt signaling pathway. In summary, these results indicate that the GLP-1 receptor agonist Exendin-4 promotes proliferation of senescent osteoblasts by up-regulating IGF-1R expression and activating the IGF-1/PI3K/Akt signaling pathway, thereby preventing senile osteoporosis. • Exendin-4 treatment enhances proliferation in senescent rat osteoblasts. • Low-dose Exendin-4 activates the IGF-1/IGF-1R pathway in senescent rat osteoblasts. • Exedin-4 reduces expression of senescence genes in high passage rat osteoblasts. [ABSTRACT FROM AUTHOR]

Insulinomas are neuroendocrine tumors arising from the pancreatic beta cells. Currently, surgical resection is the therapy of choice, and therefore, preoperative localization of insulinomas is essential. Nearly all insulinomas show overexpression of the glucagon‐like peptide‐1 receptor (GLP‐1R), and therefore, radiolabeled GLP‐1 peptide analog exendin‐4 can be used for diagnosis and preoperative localization with nuclear imaging. Here, we present an overview of the development and clinical implementation of exendin‐4–based tracers for single‐photon emission computed tomography (SPECT) and positron emission tomography (PET) imaging of insulinomas, and we address the potential use of this molecule for optical imaging. At last, we discuss the possibilities and pitfalls of the use of exendin‐4–based tracers for therapeutic applications such as peptide receptor radionuclide therapy (PRRT) or targeted photodynamic therapy (tPDT), giving a future outlook on the use of exendin‐4 in insulinoma theranostics. [ABSTRACT FROM AUTHOR]

Glucagon-like peptide-1 receptor (GLP-1R) is a kind of G protein coupled receptor which regulates the insulin secretion and serves as potential target in the diagnosis of functional pancreas neuroendocrine tumor. The aim of this study was to evaluate the feasibility of GLP-1R targeted tracer [68Ga]Ga-NOTA-MAL-Cys39-exendin-4 in the detection of insulinoma. NOTA-MAL-Cys39-exendin-4 was synthesized and then radiolabeled with gallium-68 in iQS® Ga-68 Fluidic Labeling Module. The in vitro binding affinity and cell uptake studies were evaluated in INS-1 cells. The in vivo micro-PET/CT imaging and biodistribution studies were performed on INS-1 xenograft tumor models. [68Ga]Ga-NOTA-MAL-Cys39-exendin-4 can be efficiently radiolabelled with a yield of about 85% (non-decay corrected) and radiochemical purity of >95% with a favorable stability. The molar activity was at least 145.5 GBq/μmol. The affinity (IC50) for [68Ga]Ga-NOTA-MAL-Cys39-exendin-4 was 12.99 ± 0.81 nM. Micro-PET/CT images showed intense tumor uptake with good contrast to background. Biodistribution study showed the predominant uptake was in the kidney, followed by pancreas, and the liver and spleen just showed mild uptake in the blood-pool phase with rapid clearance. At 1 h post- injection, the tumor to blood, muscle and pancreas ratios were 30.64, 40.21 and 6.46, respectively. Blocking studies showed significantly decreased tumor uptake, which further confirmed binding affinity of [68Ga]Ga-NOTA-MAL-Cys39-exendin-4 to GLP-1R. [68Ga]Ga-NOTA-MAL-Cys39-exendin-4 was easily synthesized with high yield, favorable biodistribution and high affinity to islet tumor cell, making the tracer may have great potential in the detection of GLP-1R positive tumor such as an insulinoma. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a multifactorial condition in which, along with amyloid-β (Aβ) and tau-related pathology, the synergistic activity of co-morbidity factors promote the onset and progression of the disease. Epidemiological evidence indicates that glucose intolerance, deficits in insulin secretion, or type-2 diabetes mellitus (T2DM) participate in increasing cognitive impairment or dementia risk. Insulin plays a pivotal role in the process as the hormone critically regulates brain functioning. GLP-1, the glucagon-like peptide 1, facilitates insulin signaling, regulates glucose homeostasis, and modulates synaptic plasticity. Exenatide is a synthetic GLP-1 analog employed in T2DM. However, exenatide has also been shown to affect the signaling of the brain-derived neurotrophic factor (BDNF), synaptic plasticity, and cognitive performances in animal models. In this study, we tested whether exenatide exerts neuroprotection in a preclinical AD model set to mimic the clinical complexity of the human disease. We investigated the effects of exenatide treatment in 3xTg-AD mice challenged with a high-fat diet (HFD). Endpoints of the study were variations in systemic metabolism, insulin and neurotrophic signaling, neuroinflammation, Aβ and tau pathology, and cognitive performances. Results of the study indicate that exenatide reverts the adverse changes of BDNF signaling and the neuroinflammation status of 3xTg-AD mice undergoing HFD without affecting systemic metabolism or promoting changes in cognitive performances. [ABSTRACT FROM AUTHOR]

This study analyzed the effect of miR-19b on the protective effect of Exendin-4 on islet cells in non-obese diabetic (NOD) mice. Twenty-four NOD/LT mice were randomized, according to the random number table, into a control group (4 µg/kg•day), a low-dose group (2 µg/kg•day Exendin-4), a medium-dose group (4 µg/kg•day Exendin-4) and a high-dose group (8 µg/kg•day Exendin-4) (n=6), with miR-19b expression interfered (an interference group) except for the control group. RT-qPCR was used to detect interference results and different doses of Exendin-4 were given for 8 weeks of intervention after the interference. CD4+ and CD8+ cell levels were detected by flow cytometry, IL-2 and IL-10 levels in the peripheral blood by enzyme-linked immunosorbent assay, and the apoptosis rate of islet cells in the pancreatic tissue by TUNEL. After 4 and 8 weeks of Exendin-4 intervention, mice in the high-dose group had lower blood glucose level than the medium-dose group (P<0.05). The medium-dose group had lower CD4+ cell level than the high-dose group (P<0.05), while the medium-dose group had higher CD8+ cell level than the high-dose group (P<0.05). After 8 weeks of intervention, compared with the medium-dose group, the high-dose group had lower IL-2 level (P<0.05), but higher IL-10 level (P<0.05). After 8 weeks of intervention, the medium-dose group had a higher apoptosis rate than the high-dose group (P<0.05). In conclusion, the decrease in miR-19b expression can improve the therapeutic effect of Exendin-4 on NOD, control blood glucose effectively and improve inflammatory response and immune function, as well as reduce islet cell injury. The increase in the dose of Exendin-4 can further improve its therapeutic effect on NOD. [ABSTRACT FROM AUTHOR]

Given that the search for effective pharmacotherapies for cocaine use disorder has, thus far, been fruitless, there remains a critical need for conceptually innovative approaches toward identifying new medications to treat this disease. A better understanding of the neurocircuits and neurobiological mechanisms underlying cocaine taking and seeking may identify molecular substrates that could serve as targets for novel pharmacotherapies to treat cocaine use disorder. Recent preclinical evidence suggests that glucagon-like peptide-1 (GLP-1) receptor agonists could be re-purposed to treat cocaine craving-induced relapse. This review endeavors to comprehensively summarize the current literature investigating the efficacy of GLP-1 receptor agonists in reducing the rewarding and reinforcing effects of cocaine in animal models of cocaine use disorder. The role of central endogenous GLP-1 circuits in voluntary cocaine taking and seeking is also discussed. Behavioral, neurochemical, electrophysiological and molecular biology studies indicate that central GLP-1 receptor activation functionally modulates the mesolimbic reward system and decreases addiction-like phenotypes in rodents. Overall, an emerging preclinical literature provides compelling evidence to advance GLP-1 receptor agonists into clinical trials testing the efficacy of these medications in preventing cocaine craving-induced relapse. • Central GLP-1 receptors play an important role in the reinforcing effects of cocaine. • GLP-1 receptor agonists attenuate cocaine-mediated behaviors. • Cocaine dynamically alters the endogenous central GLP-1 system. • Central GLP-1 signaling may be a novel target to treat cocaine use disorder. [ABSTRACT FROM AUTHOR]

Exendin-4 has been clinically adopted as an effective drug for treating type 2 diabetes (T2D), but its short circulation half-life in the blood requires two injections per day to maintain effective glycemic control. This significantly limits its clinical application. In this study, we developed a tannic acid/exendin-4/Fe3+ ternary nanoparticle system to provide sustained release of exendin-4 in vivo. The formation of these nanoparticles relies on TA/exendin-4 complexation and stabilization through TA-Fe3+ coordination, where the rapid reaction kinetics can benefit from efficient mixing of all three components. Adapting our recently developed flash nanocomplexation (FNC) method, we formulated nanoparticles with high encapsulation efficiency (~ 100%) of exendin-4, high payload capacity, and high degrees of uniformity and stability because the rapid turbulent mixing facilitated a homogeneous distribution of all three components in the complexation process. Intraperitoneal injection in mice showed that exendin-4 released from the nanoparticles had an AUC 7.2-fold higher than the free exendin-4 injection. Efficacy study in a T2D mouse model showed that the optimized formulation achieved a rapid reduction of the blood glucose level to the normal range within <12 h and maintained the same level for 72 h following a single intraperitoneal dose. The blood glucose level was maintained to below the therapeutic level (< 15 mmol/L) for 6 days, and the treatment led to reduced body weight with pathological and functional improvements in the kidney and liver. This tannic acid/exendin-4/Fe3+ ternary nanoparticle system holds translational potential in treating T2D, due to its improved treatment outcomes in terms of extended release of exendin-4, prolonged control of blood glucose level, reduced dosing frequency, and improved pathological indicators. • The flash nanocomplexation produces ternary nanoparticeles with excellent sizeuniformity and high encapsulation efficiency of exendin-4. • The coordination complexation between Fe3+ and tannic acid serves as additional crosslinks to stablize the nanoparticle structure. • The ternary nanoparticles release exendin-4 in a sustained manner as a result of pH-induced dissociation after intraperitoneal administration. • A single treatment with the ternary nanoparticles gives a prolonged control over the blood glucose level in a mouse type-II diabetes model. [ABSTRACT FROM AUTHOR]

Type II diabetes mellitus (T2DM) is a chronic non‐communicable disease due to abnormal insulin actions causing uncontrolled hyperglycaemia. The treatment for T2DM, for instance, metformin and incretin mimetic, mainly focuses on the restoration of insulin sensitivity and secretion. Exendin‐4 is a short incretin‐mimetic peptide consisting of 39 amino acids. It is discovered in the venom of Heloderma suspectum as a full agonist for the glucagon‐like peptide 1 (GLP‐1) receptor and produces insulinotropic effects. It is more resistant to enzymatic degradation by dipeptidyl‐peptidase‐4 and has a longer half‐life than the endogenous GLP‐1; thus, it is further developed as an incretin hormone analogue used to treat T2DM. The helical region of the peptide first interacts with the extracellular N‐terminal domain (NTD) of GLP‐1 receptor while the C‐terminal extension containing the tryptophan cage further enhances its binding affinity. After binding to the NTD of the receptor, it may cause the receptor to switch from its auto‐inhibited state of the receptor to its auto‐activated state. Exendin‐4 enhances the physiological functions of β‐cells and the up‐regulation of GLP‐1 receptors, thus reducing the plasma glucose levels. Moreover, exendin‐4 has also been found to ameliorate neuropathy, nephropathy and ventricular remodelling. The therapeutic effects of exendin‐4 have also been extrapolated into several clinical trials. Although exendin‐4 has a reasonable subcutaneous bioavailability, its half‐life is rather short. Therefore, several modifications have been undertaken to improve its pharmacokinetics and insulinotropic potency. This review focuses on the pharmacology of exendin‐4 and the structure‐function relationships of exendin‐4 with GLP‐1 receptor. The review also highlights some challenges and future directions in the improvement of exendin‐4 as an anti‐diabetic drug. [ABSTRACT FROM AUTHOR]

• The CeA lesion produces only minor disruptive effects on chow and high-surcose food intake. • Bilateral lesions of the CeA did not attenuate the chow intake suppression by i.p. exendin-4. • The CeA may contribute to the high-sucrose food intake suppression produced by i.p. exendin-4. The long-acting glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 suppresses food intake and decreases body weight. However, the mediating site(s) of action of the anorectic effects induced by peripheral administration of GLP-1R agonists are not well known. The present study investigated the effects of bilateral lesions of the central nucleus of the amygdala (CeA) on the suppression of chow and high-sucrose food intake by exendin-4 in rats. Adult male Sprague-Dawley rats with bilateral sham or electrolytic lesions (400 μA; 25 s) of the CeA were used for this experiment. No significant difference was found in the daily chow intake and high-sucrose food intake in rats with CeA lesion compared to sham-operated rats. Bilateral lesions of the CeA significantly attenuated the suppression of high-sucrose food intake by i.p. exendin-4, but not the suppression of chow intake. These results suggest that a mediating role of the CeA on the peripheral effects of exendin-4. [ABSTRACT FROM AUTHOR]

Diabetes mellitus is a metabolic disorder that results in glucotoxicity and the formation of advanced glycated end products (AGEs), which mediate several systemic adverse effects, particularly in the brain tissue. Alterations in glutamatergic neurotransmission and cognitive impairment have been reported in DM. Exendin-4 (EX-4), an analogue of glucagon-like peptide-1 (GLP-1), appears to have beneficial effects on cognition in rats with chronic hyperglycemia. Herein, we investigated the ability of EX-4 to reverse changes in AGE content and glutamatergic transmission in an animal model of DM looking principally at glutamate uptake and GluN1 subunit content of the N-methyl-d-aspartate (NMDA) receptor. Additionally, we evaluated the effects of EX-4 on in vitro models and the signaling pathway involved in these effects. We found a decrease in glutamate uptake and GluN1 content in the hippocampus of diabetic rats; EX-4 was able to revert these parameters, but had no effect on the other parameters evaluated (glycemia, C-peptide, AGE levels, RAGE, and glyoxalase 1). EX-4 abrogated the decrease in glutamate uptake and GluN1 content caused by methylglyoxal (MG) in hippocampal slices, in addition to leading to an increase in glutamate uptake in astrocyte culture cells and hippocampal slices under basal conditions. The effect of EX-4 on glutamate uptake was mediated by the phosphatidylinositide 3-kinases (PI3K) signaling pathway, which could explain the protective effect of EX-4 in the brain tissue, since PI3K is involved in cell metabolism, inhibition of apoptosis, and reduces inflammatory responses. These results suggest that EX-4 could be used as an adjuvant treatment for brain impairment associated with excitotoxicity. [ABSTRACT FROM AUTHOR]

Abstract Traumatic brain injury (TBI) is a neurodegenerative disorder for which no effective pharmacological treatment is available. Glucagon-like peptide 1 (GLP-1) analogues such as Exenatide have previously demonstrated neurotrophic and neuroprotective effects in cellular and animal models of TBI. However, chronic or repeated administration was needed for efficacy. In this study, the pharmacokinetics and efficacy of PT302, a clinically available sustained-release Exenatide formulation (SR-Exenatide) were evaluated in a concussive mild (m)TBI mouse model. A single subcutaneous (s.c.) injection of PT302 (0.6, 0.12, and 0.024 mg/kg) was administered and plasma Exenatide concentrations were time-dependently measured over 3 weeks. An initial rapid regulated release of Exenatide in plasma was followed by a secondary phase of sustained-release in a dose-dependent manner. Short- and longer-term (7 and 30 day) cognitive impairments (visual and spatial deficits) induced by weight drop mTBI were mitigated by a single post-injury treatment with Exenatide delivered by s.c. injection of PT302 in clinically translatable doses. Immunohistochemical evaluation of neuronal cell death and inflammatory markers, likewise, cross-validated the neurotrophic and neuroprotective effects of SR-Exenatide in this mouse mTBI model. Exenatide central nervous system concentrations were 1.5% to 2.0% of concomitant plasma levels under steady-state conditions. These data demonstrate a positive beneficial action of PT302 in mTBI. This convenient single, sustained-release dosing regimen also has application for other neurological disorders, such as Alzheimer's disease, Parkinson's disease, multiple system atrophy and multiple sclerosis where prior preclinical studies, likewise, have demonstrated positive Exenatide actions. Highlights • PT302 is a sustained-release formulation of Exenatide that generates steady-state plasma levels. • PT302 post-treatment mitigates neuronal loss induced by mild TBI in mice. • PT302 post-treatment mitigates neuroinflammation induced by mild TBI in mice. • PT302 post-treatment mitigates behavioral impairments induced by mild TBI in mice. • All the above was achieved at clinically translatable doses of PT302. [ABSTRACT FROM AUTHOR]

Objective. Endoplasmic reticulum stress (ERS) is suspected as an important factor in the initiation of insulin resistance. Aim. To explore the effects of exendin-4 (Ex-4) on the endoplasmic reticulum stress (ERS)-mediated insulin resistance in 3T3-L1 adipocytes. In our study, 3T3-L1 adipocytes were pre-treated with ERS inhibitors tauroursodeoxycholic acid (TUDCA), Ex-4 and an ERS inducer tunicamycin (TM) then induced insulin resistance. Glucose consumption of the adipocytes was measured. Western blots determined the protein levels of ERS markers and insulin signaling pathway. Results. TM treatment reduced insulin-stimulated glucose consumption by 19.7% in 3T3-L1 adipocytes. This repression was blunted by 24h pre-treatment with TUDCA or Ex-4. Ex-4 augmented insulin-stimulated glucose consumption in adipocytes by 14.9%. Western blotting showed that TM treatment significantly increased the ER stress markers including p-IRE, p-JNK, p-PERK, p-eIF2a and ATF6 expression, whereas 24h pre-treatment of adipocytes with TUDCA or Ex-4 alleviated the ER stress. Ex-4 alleviates ERS-induced insulin resistance by upregulating the expression of phosphorylated Akt. Conclusion. ERs mediates insulin resistance in 3T3-L1 adipocytes, and exendin-4 significantly improves this insulin resistance. [ABSTRACT FROM AUTHOR]

Organ transplantation is the gold standard therapy for the majority of patients with terminal organ failure. However, it is still a limited treatment especially due to the low number of brain death (BD) donors in relation to the number of waiting list recipients. Strategies to increase the quantity and quality of donor organs have been studied, and the administration of exendin-4 (Ex-4) to the donor may be a promising approach. Male Wistar rats were randomized into 3 groups: (1) control, without central nervous system injury; (2) BD induced experimentally, and (3) BD induced experimentally + Ex-4 administered immediately after BD induction. After BD induction, animals were monitored for 6 h before blood collection and kidney biopsy. Kidney function was assessed by biochemical quantification of plasma kidney markers. Gene and protein expressions of inflammation- and stress-related genes were evaluated by RT-qPCR and immunoblot analysis. Animals treated with Ex-4 had lower creatinine and urea levels compared with controls. BD induced oxidative stress in kidney tissue through increased expression of Ucp2, Sod2 and Inos, and Ex-4 administration reduced the expression of these genes. Ex-4 also induced increased expression of the anti-apoptotic Bcl2 gene. Nlrp3 and Tnf expressions were up-regulated in the BD group compared with controls, but Ex-4 treatment had no effect on these genes. Our findings suggest that Ex-4 administration in BD rats reduces BD-induced kidney damage by decreasing the expression of oxidative stress genes and increasing the expression of Bcl2. [ABSTRACT FROM AUTHOR]

Type 2 diabetes mellitus (T2DM) is characterized by the dysfunction and loss of pancreatic islet β-cells, in part due to islet amyloid deposits derived from islet amyloid polypeptide (IAPP). The glucagon-like peptide-1 (GLP-1) receptor agonist exendin-4 enhances the insulin secretory response by increasing β-cell mass in T2DM. However, it is unknown whether exendin-4 protects β-cells from IAPP-mediated autophagy and apoptosis. In the present study, reverse transcription-quantitative polymerase chain reaction, ELISA and western blotting were used to detected the mRNA and protein expression of insulin/hIAPP and other signaling molecules, while the mechanisms underlying these effects were also determined. Exendin-4 increased the level of insulin secretion, which was greater than that of IAPP, leading to a beneficial IAPP/insulin secretion pattern. In MIN6 cells incubated with 25 mM glucose, exendin-4 decreased the ratio of light chain 3 (LC3)-II/I, which was accompanied by an increase in p62 protein. In a hIAPP-overexpressing MIN6 cell model, exendin-4 prevented the hIAPP-induced increase in the LC3II/I ratio and decrease in p62 expression. In addition, exendin-4 pretreatment reduced hIAPP-induced activation of cleaved caspase-3, suggesting that exendin-4 may protect MIN6 cells against apoptosis. Taken together, the results highlight hIAPP as a critical mediator of β-cell loss and suggest that the GLP-1 receptor agonist exendin-4 may be a potential therapeutic agent for hIAPP-induced β-cell damage. [ABSTRACT FROM AUTHOR]

Background: Preclinical studies in rodents have demonstrated inhibitory effects of glucagon-like peptide-1 (GLP-1) receptor stimulation on alcohol consumption. The effects of GLP-1 receptor stimulation on alcohol intake in primates have not been investigated.Methods: We performed placebo-controlled studies on the effects of the GLP-1 receptor agonists exenatide and liraglutide on alcohol consumption in alcohol-preferring male African vervet monkeys. Monkeys selected for voluntary alcohol drinking were observed for at least 10 days of baseline drinking and allocated to drug or vehicle (n = 11-12 per group) balanced with respect to alcohol intake. Monkeys had access to alcohol 4 h/day. In a first study, monkeys were treated with exenatide 0.04 mg/kg or vehicle once weekly for 5 weeks to obtain steady-state plasma levels. In a second study, monkeys were treated daily with liraglutide (increasing dosing, 10 to 50 μg/kg/day) or vehicle over 2 weeks. In both studies, access to alcohol was suspended during drug up-titration. Then, alcohol was again made available 4 h/day and treatment was continued for 2 weeks, during which alcohol intake was recorded. Observation of alcohol intake was continued for a week of drug washout.Results: Liraglutide and to a lesser extent exenatide significantly reduced alcohol consumption without causing any signs of emesis and with no effect on water intake as compared to vehicle.Conclusions: The present study demonstrates for the first time that GLP-1 receptor agonists can reduce voluntary alcohol drinking in non-human primates. The data substantiate the potential usefulness of GLP-1 receptor agonists in the treatment of alcohol use disorder. [ABSTRACT FROM AUTHOR]

Radiolabelled ligands of glucagon-like peptide 1 receptor (GLP-1R) have been used to image the GLP-1R-expressing tissues (e.g., islets and insulinoma). Here, we introduced human glucagon-like peptide 1 receptor (hglp-1r) gene as a novel radionuclide reporter gene to broaden its applications in molecular imaging in vivo. Transient and stable baculoviral vectors (BV) were re-constructed and used to transfer the hglp-1r gene or enhanced green fluorescent protein (egfp) reporter gene into the stem cells or tumor cells. Cell proliferation assay and flow cytometry analysis demonstrated that BV-mediated reporter gene transferring and expression was biosafe and highly efficient. The BV-mediated exogenous hGLP-1R in target cells showed same ligand-receptor binding characteristics compared with its counterpart in insulinoma cells. Furthermore, the ligand-receptor binding assay showed a high affinity (IC50 = 0.3708 nM) and robust correlation (R2 = 0.9264) between the fluorescein-tagged or radiolabeled ligand probes and exogenous hGLP-1R in target cells. The target cells transferred with BV-mediated hGLP-1R could be clearly visualized in nude mice by micro-PET, which was capable of the purposes of short-term tracking transplanted stem cells or long-term monitoring tumor formation. Then, the image-based analysis and bio-distribution analysis quantitatively confirmed high target-to-background ratio of hGLP-1R-expressing cells. This study also investigated the endogenous GLP-1R-expressing organs/tissues in nude mice in the hGLP-1R radionuclide reporter gene imaging. Summarily, we evaluated the utility of hglp-1r gene as a novel radionuclide reporter gene, and demonstrated that it was a favorable and promising candidate of molecular imaging tool, which would expand the spectrum of radionuclide reporter gene imaging systems. [ABSTRACT FROM AUTHOR]

Abstract 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. Highlights • c-fos analyzed by iDISCO immunostaining and 3D imaging gave the unique possibility to study global brain c-fos reactivity. • Ex4 alone induced significant c-fos immunoreactivity in 9 brain regions and no effect by PYY(3-36) compared to vehicle. • PYY(3-36) and Ex4 in combination induced significant c-fos immunoreactivity in 25 brain regions compared to vehicle. • Within the mediobasal ARH and AP/NTS/PB/CeA anorectic pathway a synergistic c-fos response was found by PYY(3-36) and Ex4. • PYY(3-36) and Ex4 also activated LRNm and SUT which not previously has been reported in the regulation of energy homeostasis. [ABSTRACT FROM AUTHOR]

Abstract Exendin-4 is a glucagon-like peptide-1 (GLP-1) receptor agonist and potent insulinotropic agent for type 2 diabetes patients; however, its therapeutic utility is limited due to the frequent injections required. Long-acting agonists reduce the number of injections, but they can compromise potency. In this study, chondroitin sulfate-g-glycocholic acid-coated and Exendin-4 (Ex-4)-loaded liposomes (EL-CSG) were prepared for oral administration of Ex-4. The Ex-4 loading efficiency was 77% and the loading content in the nanoparticles was 1 wt-%. In rat models, a single oral dose (200 μg/kg) of EL-CSG showed a relative oral bioavailability of 19.5%, compared with subcutaneous administration (20 μg/kg), and sustained pharmacokinetics for up to 72 h. The overall long-term pharmacodynamic effects, assessed by hemoglobin A1c (HbA1c), body weight, and blood lipid concentrations, of daily oral EL-CSG (300 μg/kg) for four weeks were equivalent to or better than daily subcutaneous injections of free Ex-4 solution (20 μg/kg). Graphical abstract Unlabelled Image [ABSTRACT FROM AUTHOR]

Key points: The pancreatic islets of Langerhans maintain glucose homeostasis through insulin secretion, where insulin secretion dynamics are regulated by intracellular Ca2+ signalling and electrical coupling of the insulin producing β‐cells in the islet.We have previously shown that cytokines decrease β‐cell coupling and that compounds which increase cAMP can increase coupling.In both mouse and human islets exendin‐4, which increases cAMP, protected against cytokine‐induced decreases in coupling and in mouse islets preserved glucose‐stimulated calcium signalling by increasing connexin36 gap junction levels on the plasma membrane.Our data indicate that protein kinase A regulates β‐cell coupling through a fast mechanism, such as channel gating or membrane organization, while Epac2 regulates slower mechanisms of regulation, such as gap junction turnover.Increases in β‐cell coupling with exendin‐4 may protect against cytokine‐mediated β‐cell death as well as preserve insulin secretion dynamics during the development of diabetes. The pancreatic islets of Langerhans maintain glucose homeostasis. Insulin secretion from islet β‐cells is driven by glucose metabolism, depolarization of the cell membrane and an influx of calcium, which initiates the release of insulin. Gap junctions composed of connexin36 (Cx36) electrically couple β‐cells, regulating calcium signalling and insulin secretion dynamics. Cx36 coupling is decreased in pre‐diabetic mice, suggesting a role for altered coupling in diabetes. Our previous work has shown that pro‐inflammatory cytokines decrease Cx36 coupling and that compounds which increase cAMP can increase Cx36 coupling. The goal of this study was to determine if exendin‐4, which increases cAMP, can protect against cytokine‐induced decreases in Cx36 coupling and altered islet function. In both mouse and human islets, exendin‐4 protected against cytokine‐induced decreases in coupling and preserved glucose‐stimulated calcium signalling. Exendin‐4 also protected against protein kinase Cδ‐mediated decreases in Cx36 coupling. Exendin‐4 preserved coupling in mouse islets by preserving Cx36 levels on the plasma membrane. Exendin‐4 regulated Cx36 coupling via both protein kinase A (PKA)‐ and Epac2‐mediated mechanisms in cytokine‐treated islets. In mouse islets, modulating Epac2 had a greater impact in mediating Cx36 coupling, while in human islets modulating PKA had a greater impact on Cx36 coupling. Our data indicate that PKA regulates Cx36 coupling through a fast mechanism, such as channel gating, while Epac2 regulates slower mechanisms of regulation, such as Cx36 turnover in the membrane. Increases in Cx36 coupling with exendin‐4 may protect against cytokine‐mediated β‐cell dysfunction to insulin secretion dynamics during the development of diabetes. Key points: The pancreatic islets of Langerhans maintain glucose homeostasis through insulin secretion, where insulin secretion dynamics are regulated by intracellular Ca2+ signalling and electrical coupling of the insulin producing β‐cells in the islet.We have previously shown that cytokines decrease β‐cell coupling and that compounds which increase cAMP can increase coupling.In both mouse and human islets exendin‐4, which increases cAMP, protected against cytokine‐induced decreases in coupling and in mouse islets preserved glucose‐stimulated calcium signalling by increasing connexin36 gap junction levels on the plasma membrane.Our data indicate that protein kinase A regulates β‐cell coupling through a fast mechanism, such as channel gating or membrane organization, while Epac2 regulates slower mechanisms of regulation, such as gap junction turnover.Increases in β‐cell coupling with exendin‐4 may protect against cytokine‐mediated β‐cell death as well as preserve insulin secretion dynamics during the development of diabetes. [ABSTRACT FROM AUTHOR]

Highlights • Exendin-4 increases phosphorylation of cofilin and promotes actin cytoskeleton rearrangement. • Exendin-4 protects SH-SY5Y and PC12 cells from Nogo-A-Δ20-mediated spreading inhibition. • Exendin-4 induces SH-SY5Y differentiation and reduces Nogo-A-Δ20-mediated growth cone collapse. • Exendin-4 significantly reduce SH-SY5Y cell migration. • Exendin-4 decreases the expression and activation of RhoA/Rock1 via PI3K signaling pathway. Abstract Exendin-4 is a protein of the GLP-1 family currently used to treat diabetes. Recently, a greater number of biological properties have been associated with the GLP-1 family. Our data shows that exendin-4 treatment significantly increases the cytoskeleton rearrangement, which leads to an increasingly differentiated phenotype and reduced cell migration. We also found that exendin-4 could prevent SH-SY5Y and PC12 cells from Nogo-A-Δ20 mediated spreading inhibition and neurite collapse. Western blot analysis indicated that exendin-4 treatment both reduced the expression and activation of RhoA via the PI3K signaling pathway. These data suggest that exendin-4 may protect nerve regeneration by preventing the inhibition of Nogo-A via down-regulating RhoA expression and activation. [ABSTRACT FROM AUTHOR]

Highlights • Glucagons and oxyntomodulins from phylogenetic ancient fish stimulate insulin release from clonal β-cells and mouse islets. • Insulinotropic activities of paddlefish and lamprey glucagons were attenuated by a GLP1R antagonist and a GCGR antagonist. • Paddlefish and lamprey glucagon stimulate cAMP production in cells transfected with GLP1R and also with GCGR. • Administration of the fish peptides improved glucose tolerance and stimulated insulin release in mice. Abstract The insulinotropic and antihyperglycaemic properties of glucagons from the sea lamprey (Petromyzontiformes), paddlefish (Acipenseriformes) and trout (Teleostei) and oxyntomodulin from dogfish (Elasmobranchii) and ratfish (Holocephali) were compared with those of human glucagon and GLP-1 in mammalian test systems. All fish peptides produced concentration-dependent stimulation of insulin release from BRIN-BD11 rat and 1.1 B4 human clonal β-cells and isolated mouse islets. Paddlefish glucagon was the most potent and effective peptide. The insulinotropic activity of paddlefish glucagon was significantly (P < 0.01) decreased after incubating BRIN-BD11 cells with the GLP1R antagonist, exendin-4(9-39) and the GCGR antagonist [des-His1,Pro4, Glu9] glucagon amide but GIPR antagonist, GIP(6-30)Cex-K40[palmitate] was without effect. Paddlefish and lamprey glucagons and dogfish oxyntomodulin (10 nmol L−1) produced significant (P < 0.01) increases in cAMP concentration in Chinese hamster lung (CHL) cells transfected with GLP1R and human embryonic kidney (HEK293) cells transfected with GCGR. The insulinotropic activity of paddlefish glucagon was attenuated in CRISPR/Cas9-engineered GLP1R knock-out INS-1 cells but not in GIPR knock-out cells. Intraperitoneal administration of all fish peptides, except ratfish oxyntomodulin, to mice together with a glucose load produced significant (P < 0.05) decreases in plasma glucose concentrations and paddlefish glucagon produced a greater release of insulin compared with GLP-1. Paddlefish glucagon shares the sequences Glu15-Glu16 and Glu24-Trp25-Leu26-Lys27-Asn28-Gly29 with the potent GLP1R agonist, exendin-4 so may be regarded as a naturally occurring, dual-agonist hybrid peptide that may serve as a template design of new drugs for type 2 diabetes therapy. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Conjugation of tauroursodeoxycholic acid (TUDCA) to Lys-27 of exendin-4 (Ex4). • TUDCA-Lys27-Ex4 (TUM1-Ex4) showed strong activity as a GLP-1 receptor agonist. • TUM1-Ex4 reduced the expression of GRP78 and levels of phosphorylated eIF2α. Abstract Endoplasmic reticulum stress has been considered a major cause of pancreatic β-cell dysfunction and apoptosis leading to diabetes. Glucagon-like peptide-1 receptor activation and chemical chaperones have been known to reduce endoplasmic reticulum stress and improve β-cell function and survival. The purpose of this study was to prepare and evaluate the chemical chaperone tauroursodeoxycholic acid-conjugated exendin-4 as a protective agent for pancreatic β-cells. Mono-tauroursodeoxycholic acid-Lys27-exendin-4 conjugate (TUM1-Ex4) showed better receptor binding affinity than other conjugates with strong in vitro insulinotropic activity in rat pancreatic β-cells and in vivo hypoglycemic activity in type 2 diabetic db/db mice. In INS-1 cells under endoplasmic reticulum stress induced by thapsigargin, TUM1-Ex4 promoted cell survival in a dose-dependent manner. In western blot analysis, TUM1-Ex4 reduced the expression of the endoplasmic reticulum stress marker GRP78 and phosphorylation of the translation initiation factor eIF2α. These results reveal that TUM1-Ex4 accelerates translational recovery and contributes to β-cell protection and survival. The present study indicates that the chemical chaperone-coupled glucagon-like peptide-1 receptor agonist is a feasible therapeutic strategy to enhance β-cell function and survival. [ABSTRACT FROM AUTHOR]

Abstract Activation of endoplasmic reticulum (ER) stress is involved in the development of nonalcoholic fatty liver disease. Glucagon-like peptide-1 (GLP-1) has been reported to reduce hepatic steatosis, but the underlying mechanism has not been fully elucidated. Here, we investigated whether exendin-4 (EX-4), a GLP-1 receptor analogue, improves hepatic steatosis through ER stress reduction. Furthermore, we explored which ER stress pathway is involved in this process, with a focus on the protein kinase RNA-like ER kinase (PERK)-nuclear factor erythroid-derived 2-related factor 2 (Nrf2) pathway. EX-4 treatment reduced hepatic lipid accumulation by suppressing the expression of lipogenic genes and restoring the expression of β-oxidation genes in palmitate-treated HepG2 cells and high fat diet (HFD)-fed mice. In addition, EX-4 treatment suppressed hepatic ER stress activation in HFD-fed mice and tunicamycin-treated mice. In particular, EX-4 treatment restored HFD- and tunicamycin-induced Nrf2 nuclear translocation to control levels. Inhibition of Nrf2 by siRNA enhanced phosphorylation of PERK and eukaryotic translation initiation factor 2α (eIF2α), as well as other substrates of the PERK pathway. Nrf2 knockdown also inhibited the protective effects of EX-4 against lipid accumulation, ER stress activation, and cell death in palmitate-treated HepG2 cells. EX-4 treatment prevents hepatic steatosis and improves cell survival by regulating hepatic lipid metabolism and reducing ER stress activation, and Nrf2 plays an essential role in the protective effect of GLP-1 on hepatic steatosis. Highlights • GLP-1 receptor agonist prevents hepatic steatosis by regulating hepatic lipid metabolism. • GLP-1 receptor agonist reduces endoplasmic reticulum stress induced by saturated fatty acids and high fat diet. • Nrf2 plays an essential role in the protective effect of GLP-1 receptor agonist on hepatic steatosis. [ABSTRACT FROM AUTHOR]

Mitochondrial dysfunction under diabetic condition leads to the development and progression of neurodegenerative complications. Recently, the beneficial effects of glucagon-like peptide-1 (GLP-1) receptor agonists on diabetic neuropathies have been widely recognized. However, molecular mechanisms underlying the neuroprotective effects of GLP-1 receptor agonists against high glucose (HG)-induced neuronal damages is not completely elucidated. Here, we investigated the underlying mechanisms of GLP-1 receptor agonist treatment against oxidative stress, mitochondrial dysfunction, and neuronal damages under HG conditions mimicking a diabetic hyperglycemic state in SH-SY5Y neuroblastoma cells. We revealed that treatment with exendin-4, a GLP-1 receptor agonist, not only increased the expression of survival markers, phospho-Akt/Akt and Bcl-2, but also decreased the expression of pro-apoptotic marker, Bax, and reduced the levels of reactive oxygen species (ROS) defense markers (catalase, SOD-2, and HO-1) under HG conditions. The expressions of mitochondrial function associated genes, MCU and UCP3, and mitochondrial fission genes, DRP1 and FIS1, were decreased by exendin-4 compared to non-treated levels, while the protein expression levels of mitochondrial homeostasis regulators, Parkin and PINK1, were enhanced. In addition, blockade of Epac and Akt activities was able to antagonize these neuroprotective effects of exendin-4. Collectively, we demonstrated that stimulation of GLP-1 receptor propagates a neuroprotective cascade against the oxidative stress and mitochondrial dysfunction as well as augments survival through the Epac/Akt-dependent pathway. Therefore, the revealed mechanisms underlying GLP-1 receptor pathway by preserving mitochondrial homeostasis would be a therapeutic candidate to alleviate neuronal dysfunctions and delay the progression of diabetic neuropathies. [Display omitted] • Neuroprotective effects of exendin-4 occurred through GLP-1R/Epac/Akt signaling. • Exendin-4 induced survival (p-Akt; Bcl-2) and decreased pro-apoptotic (Bax) markers. • Exendin-4 decreased mitochondrial indicators (MCU; UCP3; DRP1; FIS1). • Mitochondrial homeostasis regulators (Parkin; PINK1) were enhanced by exendin-4. [ABSTRACT FROM AUTHOR]

PET/CT imaging of glucagon-like peptide receptor 1 has recently filled a gap in reliably diagnosing insulinoma through non-invasive means. 68Ga-labelled derivatives of exendin-4 show high sensitivity as well as sufficient serum stability to enable routine clinical application. Here, we provide data for automated production of [68Ga][Nle14,Lys40(Ahx-DOTA-Ga)NH 2 ]exendin-4 ([68Ga]Ga-DOTA-exendin-4) on a cassette based synthesis module (Modular-Lab PharmTracer, Eckert & Ziegler) using commercially available cassettes in combination with an approved 68Ge/68Ga generator (GalliaPharm, Eckert & Ziegler). This setup ensured high reproducibility as well as low radiation burden for the production team. Quality control including determination of radiochemical purity was performed by RP-HPLC using a water/0.1 % TFA/acetonitrile gradient on a C18 column. A modified TLC system with ammonium acetate & methanol as mobile phase and a novel limit test for determination of polysorbate 80 content in the final formulation are also described in this study. Reliable yields as well as high molar activity for patient use were only achieved using a fractionated elution approach. Batch data showed radiochemical purity of >93 % as determined by RP-HPLC and TLC as well as good stability over 2 h post production. Testing for polysorbate 80 confirmed a concentration <1 mg/mL in the final product solution. Specifications for routine production were established based on existing Pharmacopeia monographs for other radiopharmaceuticals and were validated with 5 master batches. The described synthesis method enables reproducible, automated in-house production of [68Ga]Ga-DOTA-exendin-4 for routine clinical application. [Display omitted] [ABSTRACT FROM AUTHOR]

Protective effects of exendin-4 (glucagon-like peptide-1 -GLP-1- receptor agonist) and des-fluoro-sitagliptin (dipeptidyl peptidase-4 inhibitor) on fructose-induced hepatic disturbances were evaluated in prediabetic rats. Complementary, a possible direct effect of exendin-4 in human hepatoblastoma-derived cell line HepG2 incubated with fructose in presence/absence of exendin-9-39 (GLP-1 receptor antagonist) was investigated. In vivo, after 21 days of fructose rich diet, we determined: glycemia, insulinemia, and triglyceridemia; hepatic fructokinase, AMP-deaminase, and G-6-P dehydrogenase (G-6-P DH) activities; carbohydrate-responsive element-binding protein (ChREBP) expression; triglyceride content and lipogenic gene expression (glycerol-3-phosphate acyltransferase -GPAT-, fatty acid synthase -FAS-, sterol regulatory element-binding protein-1c -SREBP-1c); oxidative stress and inflammatory markers expression. In HepG2 cells we measured fructokinase activity and triglyceride content. Hypertriglyceridemia, hyperinsulinemia, enhanced liver fructokinase, AMP-deaminase, and G-6-P DH activities, increased ChREBP and lipogenic genes expression, enhanced triglyceride level, oxidative stress and inflammatory markers recorded in fructose fed animals, were prevented by co-administration of either exendin-4 or des-fluoro-sitagliptin. Exendin-4 prevented fructose-induced increase in fructokinase activity and triglyceride contain in HepG2 cells. These effects were blunted co-incubating with exendin-9-39. The results demonstrated for the first time that exendin-4/des-fluro-sitagliptin prevented fructose-induced endocrine-metabolic oxidative stress and inflammatory changes probably acting on the purine degradation pathway. Exendin 9–39 blunted in vitro protective exendin-4 effects, thereby suggesting a direct effect of this compound on hepatocytes through GLP-1 receptor. Direct effect on fructokinase and AMP-deaminase activities, with a key role in the pathogenesis of liver dysfunction induced by fructose, suggests purine degradation pathway constitute a potential therapeutic objective for GLP-1 receptor agonists. • Purine degradation pathway is a potential new therapeutic target of incretins. • Protective effect is mediated by direct action on GLP-1 receptors in HepG2 cells. • New therapeutic targets serve to prevent transition from pre-diabetes to T2D. [ABSTRACT FROM AUTHOR]

Glucagon-like peptide 1 (GLP-1) plays an important role in regulating bone remodeling, and GLP-1 receptor agonist shows a positive relationship with osteoblast activity. However, GLP-1 receptor is not found in osteoblast, and the mechanism of GLP-1 receptor agonist on regulating bone remodeling is unclear. Here, we show that the GLP-1 receptor agonist exendin-4 (Ex-4) promoted bone formation and increased bone mass and quality in a rat unloading-induced bone loss model. These functions were accompanied by an increase in osteoblast number and serum bone formation markers, while the adipocyte number was decreased. Furthermore, GLP-1 receptor was detected in bone marrow stromal cells (BMSCs), but not in osteoblast. Activation of GLP-1 receptor by Ex-4 promoted the osteogenic differentiation and inhibited BMSC adipogenic differentiation through regulating PKA/β-catenin and PKA/PI3K/AKT/GSK3β signaling. These findings reveal that GLP-1 receptor regulates BMSC osteogenic differentiation and provide a molecular basis for therapeutic potential of GLP-1 against osteoporosis.

Selective MSCs differentiation protocol into pancreatic beta cells was conducted in the present study using exendin-4 and TGF-beta. Differentiated and undifferentiated MSCs were assessed in experimental type I diabetes in rats. Ninety female white albino rats were included in the study and divided equally (n=15/group) into 6 groups: healthy control, healthy control rats received acellular tissue culture medium, diabetic rats, diabetic rats received acellular tissue culture medium, diabetic rats received undifferentiated MSCs and diabetic rats received differentiated MSCs. Therapeutic efficacy of undifferentiated versus differentiated MSCs was evaluated via assessment of quantitative gene expressions of insulin1, insulin 2, Smad-2, Smad-3, PDX-1, PAX-4, neuroD. Blood glucose and insulin hormone levels were also assessed. Results showed that quantitative gene expressions of all studied genes showed significant decrease in diabetic rat groups. Use of undifferentiated and differentiated MSCs led to a significant elevation of expression levels of all genes with more superior effect with differentiated MSCs except smad-2 gene. As regards insulin hormone levels, use of either undifferentiated or differentiated MSCs led to a significant elevation of its levels with more therapeutic effect with differentiated MSCs. Blood glucose levels were significantly decreased with both undifferentiated and differentiated MSCs in comparison to diabetic groups but its levels were normalized 2 months after injection of differentiated MSCs. In conclusion, use of undifferentiated or differentiated MSCs exhibited significant therapeutic potentials in experimental type I diabetes in rats with more significant therapeutic effect with the use of differentiated MSCs.

Objective: To evaluate the effect of Exendine-4 (EX-4), a Glucagon-like peptide 1 (GLP-1) receptor agonist, on the differentiation of insulin-secreting cells (IPCs) from rat adipose-derived mesenchymal stem cells(ADMSCs). Materials and Methods: In this experimental study, ADMSCs were isolated from rat adipose tissue and exposed to induction media with or without EX-4. After induction, the existence of IPCs was confirmed by morphology analysis, expression pattern analysis of islet-specific genes (Pdx-1, Glut-2 and Insulin) and insulin synthesis and secretion. Results: IPCs induced in presence of EX-4 were morphologically similar to pancreatic islet-like cells. Expression of Pdx-1, Glut-2 and Insulin genes in EX-4 treated cells was significantly higher than the cells exposed to differentiation media without EX-4. Compared to EX-4 untreated ADMSCs, insulin release from EX-4 treated ADMSCs showed a nearly 2.5 fold (P

This review article discusses the preclinical evidence and clinical trials testing the use of a peptide agonist of the glucagon-like peptide (GLP) receptor that promotes insulin secretion in the animal models of and patient with Parkinson's disease (PD). In particular, we focus on the therapeutic effects of the GLP receptor agonist exendin-4, also called exenatide, in PD. The ultimate goal of this article is to provide a critical assessment of the laboratory and clinical data toward guiding the translation of exendin-4 as a clinically relevant therapeutic for PD. [ABSTRACT FROM AUTHOR]

Glucagon-like peptide 1 (GLP-1) mimetics have been approved as an adjunct therapy for glycemic control in type 2 diabetic patients for the increased insulin secretion under hyperglycemic conditions. Recently, it is reported that such agents elicit neuroprotective effects against diabetes-associated cognitive decline. However, there is an issue of poor compliance by multiple daily subcutaneous injections for sufficient glycemic control due to their short duration, and neuroprotective actions were not fully studied, yet. In this study, using the prepared exendin-4 fusion protein agent, we investigated the pharmacokinetic profile and the role of this GLP-1 mimetics on memory deficits in a high-fat diet (HFD)/streptozotocin (STZ) mouse model of type 2 diabetic mellitus. After induction of diabetes, mice were administered weekly by intraperitoneal injection of GLP-1 mimetics for 6 weeks. This treatment reversed HFD/STZ-induced metabolic symptoms of increased body weight, hyperglycemia, and hepatic steatosis. Furthermore, the impaired cognitive performance of diabetic mice was significantly reversed by GLP-1 mimetics. GLP-1 mimetic treatment also reversed decreases in GLP-1/GLP-1 receptor expression levels in both the pancreas and hippocampus of diabetic mice; increases in hippocampal inflammation, mitochondrial fission, and calcium-binding protein levels were also reversed. These findings suggest that GLP-1 mimetics are promising agents for both diabetes and neurodegenerative diseases that are associated with increased GLP-1 expression in the brain.

BackgroundSelenoprotein P (SEPP1) and fetuin-A, both circulating liver-derived glycoproteins, are novel biomarkers for insulin resistance and nonalcoholic fatty liver disease. However, the effect of exendin-4 (Ex-4), a glucagon-like peptide-1 receptor agonist, on the expression of hepatokines, SEPP1, and fetuin-A, is unknown.MethodsThe human hepatoma cell line HepG2 was treated with palmitic acid (PA; 0.4 mM) and tunicamycin (tuni; 2ug/ml) with or without exendin-4 (100 nM) for 24 hours. The change in expression of PA-induced SEPP1, fetuin-A, and endoplasmic reticulum (ER) stress markers by exendin-4 treatment were evaluated using quantitative real-time reverse transcription polymerase chain reaction and Western blotting. Transfection of cells with AMP-activated protein kinase (AMPK) small interfering RNA (siRNA) was performed to establish the effect of exendin-4-mediated AMPK in the regulation of SEPP1 and fetuin-A expression.ResultsExendin-4 reduced the expression of SEPP1, fetuin-A, and ER stress markers including PKR-like ER kinase, inositol-requiring kinase 1α, activating transcription factor 6, and C/EBP homologous protein in HepG2 cells. Exendin-4 also reduced the expression of SEPP1 and fetuin-A in cells treated with tunicamycin, an ER stress inducer. In cells treated with the AMPK activator 5-aminoidazole-4-carboxamide ribonucleotide (AICAR), the expression of hepatic SEPP1 and fetuin-A were negatively related by AMPK, which is the target of exendin-4. In addition, exendin-4 treatment did not decrease SEPP1 and fetuin-A expression in cells transfected with AMPK siRNA.ConclusionThese data suggest that exendin-4 can attenuate the expression of hepatic SEPP1 and fetuin-A via improvement of PA-induced ER stress by AMPK.

Background/Aims: Prevention of diabetes requires maintenance of a functional beta-cell mass, the postnatal growth of which depends on beta cell proliferation. Past studies have shown evidence of an effect of an incretin analogue, Exendin-4, in promoting beta cell proliferation, whereas the underlying molecular mechanisms are not completely understood. Methods: Here we studied the effects of Exendin-4 on beta cell proliferation in vitro and in vivo through analysing BrdU-incorporated beta cells. We also analysed the effects of Exendin-4 on beta cell mass in vivo, and on beta cell number in vitro. Then, we applied specific inhibitors of different signalling pathways and analysed their effects on Exendin-4-induced beta cell proliferation. Results: Exendin-4 increased beta cell proliferation in vitro and in vivo, resulting in significant increases in beta cell mass and beta cell number, respectively. Inhibition of PI3K/Akt signalling, but not inhibition of either ERK/MAPK pathway, or JNK pathway, significantly abolished the effects of Exendin-4 in promoting beta cell proliferation. Conclusion: Exendin-4 promotes beta cell proliferation via PI3k/Akt signaling pathway.

Diabetic retinopathy (DR) is a complication of diabetes and a leading cause of blindness in adults. Studies have shown that glucagon-like peptide-1 (GLP-1) exerts a protective effect on patients with DR. Here, we investigated the protective effects of Exendin-4, a GLP-1 analogue, on DR. We established a high-glucose-induced HREC cell model and an STZ-induced rat DR Model to study the effect of Exendin-4 in DR in vitro and in vivo . The qRT-PCR, CCK-8, TUNEL, western blotting, tube formation assays, and ELISA were performed. In addition, we overexpressed TGFB2 to observe whether the protective effect of Exendin-4 was reversed. Our results showed that Exendin-4 inhibited the progression of DR. Furthermore, the protective effect of Exendin-4 was suppressed in cells overexpressing TGFB2. Our findings suggest that Exendin-4 may be involved in the regulation of TGFB2 expression levels to inhibit DR. These results indicate that Exendin-4 could be an effective therapy for DR.

Type 2 Diabetes Mellitus (T2D) is a chronic, obesity-related, and inflammatory disorder characterize by insulin resistance, inadequate insulin secretion, hyperglycemia, and excessive glucagon secretion. Exendin-4 (EX), a clinically established antidiabetic medication that acts as a glucagon-like peptide-1 receptor agonist, is effective in lowering glucose levels and stimulating insulin secretion while significantly reducing hunger. However, the requirement for multiple daily injections due to EX's short half-life is a significant limitation in its clinical application, leading to high treatment costs and patient inconvenience. To address this issue, an injectable hydrogel system is developed that can provide sustained EX release at the injection site, reducing the need for daily injections. In this study, the electrospray technique is examine to form EX@CS nanospheres by electrostatic interaction between cationic chitosan (CS) and negatively charged EX. These nanospheres are uniformly dispersed in a pH-temperature responsive pentablock copolymer, which forms micelles and undergoes sol-to-gel transition at physiological conditions. Following injection, the hydrogel gradually degraded, exhibiting excellent biocompatibility. The EX@CS nanospheres are subsequently released, maintaining therapeutic levels for over 72 h compared to free EX solution. The findings demonstrate that the pH-temperature responsive hydrogel system containing EX@CS nanospheres can be a promising platform for the treatment of T2D., (© 2023 Wiley-VCH GmbH.)

Background: Peptidic radiotracers are preferentially excreted through the kidneys, which often results in high persistent renal retention of radioactivity, limiting or even preventing therapeutic clinical translation of these radiotracers. Exendin-4, which targets the glucagon-like-peptide 1 receptor (GLP-1R) overexpressed in insulinomas and in congenital hyperinsulinism, is an example thereof. The use of the tripeptide MVK, which is readily cleaved between methionine and valine by neprilysin at the renal brush border membrane, already showed promising results in reducing kidney uptake as reported in the literature. Based on our previous findings we were interested how linker variants with multiple copies of the MV-motive influence renal washout of radiolabelled exendin-4., Results: Three exendin-4 derivatives, carrying either one MVK, a MV-MVK or a MVK-MVK linker were synthesized and compared to a reference compound lacking a cleavable linker. In vivo results of a biodistribution in GLP-1R overexpressing tumour bearing mice at 24 h post-injection demonstrated a significant reduction (at least 57%) of renal retention of all 111 In-labeled exendin-4 compounds equipped with a cleavable linker compared to the reference compound. While the insertion of the single linker MVK led to a reduction in kidney uptake of 70%, the dual approach with the linker MV-MVK slightly, but not significantly enhanced this effect, with 77% reduction in kidney uptake compared to the reference. In vitro IC 50 and cell uptake studies were conducted and demonstrated that though the cleavable linkers negatively influenced the affinity towards the GLP-1R, cell uptake remained largely unaffected, except for the MV-MVK cleavable linker conjugate, which displayed lower cell uptake than the other compounds. Importantly, the tumour uptake in the biodistribution study was not significantly affected with 2.9, 2.5, 3.2 and 1.5% iA/g for radiolabelled Ex4, MVK-Ex4, MV-MVK-Ex4 and MVK-MVK-Ex4, respectively., Conclusion: Cleavable linkers are highly efficient in reducing the radioactivity burden in the kidney. Though the dual linker approach using the instillation of MV-MVK or MVK-MVK between exendin-4 and the radiometal chelator did not significantly outperform the single cleavable linker MVK, further structural optimization or the combination of different cleavable linkers could be a stepping stone in reducing radiation-induced nephrotoxicity., (© 2023. Springer Nature Switzerland AG.)

Highlights • GLP-1 agonists are diabetes drugs with neuroprotective effects. • The distribution of GLP-1R in the brain is associated with neurological disorders. • Aged rats have lower expression of GLP-1R compared with normal rats. • The change of GLP-1R expression in aged rats can be detected using microPET. • The results of biodistribution and IHC were consistent with microPET. Abstract The glucagon-like peptide-1 receptor (GLP-1R) has been demonstrated as a potential therapeutic target for some neurological diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and stroke. Besides, its distribution and density in brain regions are closely associated with cognition, motor function, learning and memory. Given the relationship between age and these neurological diseases, we firstly examined the influences of age on GLP-1R expression using [18F]AlF-NOTA-MAL-Cys39-exendin-4 microPET imaging. The image showed that GLP-1R expression in nearly all regions of the brain of aged rats was evidently lower than that of normal rats. Significant differences were found in olfactory, striatum, hypothalamus, substantial nigra, and hippocampus, which have inseparable relations with some mental and neurological diseases such as PD and AD. Data obtained from biodistribution and immunohistochemistry staining also confirmed the image results. Taken together, these results illustrated decreased expression of GLP-1R in the brain of aged rats can be detected by [18F]AlF-NOTA-MAL-Cys39-exendin-4, which implied GLP-1R as a reliable target and GLP-1R PET imaging could be a promising technology in the field of neurological diseases. [ABSTRACT FROM AUTHOR]

Protein transduction domains (PTDs) have been shown to promote the delivery of therapeutic proteins or peptides into the living cells. In a previous study, we showed that the double mutant of TCTP-PTD 13, TCTP-PTD 13M2, was more effective in the delivery of insulin than the wild-type TCTP-PTD 13. In this study, we applied this approach to the nasal delivery of a different peptide, exendin-4, using as carriers, several modified TCTP-PTDs, such as TCTP-PTD 13M1, 13M2, and 13M3. Nasal co-administration of TCTP-PTD 13M2 with exendin-4 showed the highest exendin-4 uptake among the three analogs in normal rats, and also decreased blood glucose levels by 43.3% compared with that of exendin-4 alone and by 18.6% compared with that of exendin-4 plus TCTP-PTD 13 in diabetic mice. We also designed an additional covalently linked conjugate of TCTP-PTD 13M2 and exendin-4 and evaluated its hypoglycemic effect after subcutaneous or intranasal delivery. Subcutaneous administration of exendin-4 that its C-terminus is covalently linked to TCTP-PTD 13M2 showed hypoglycemic effect of 42.2% compared to that in untreated group, whereas intranasal delivery was not successful in diabetic mice. We conclude that a simple mixing TCTP-PTD 13M2 with peptide/protein drugs can be potentially a generally applicable approach for intranasal delivery into animals. [ABSTRACT FROM AUTHOR]