GLP-1 receptor agonist attenuates tubular cell ferroptosis in diabetes via enhancing AMPK-fatty acid metabolism pathway through macropinocytosis.

Kidney tubules are mostly responsible for pathogenesis of diabetic kidney disease. Actively reabsorption of iron, high rate of lipid metabolism and exposure to concentrated redox-active compounds constructed the three main pillars of ferroptosis in tubular cells. However, limited evidence has indicated that ferroptosis is indispensable for diabetic tubular injury. Glucagon-like peptide-1 receptor agonist (GLP-1RA) processed strong benefits on kidney outcomes in people with diabetes. Moreover, GLP-1RA may have additive effects by improving dysmetabolism besides glucose control and weight loss. Therefore, the present study aimed at exploring the benefits of exendin-4, a high affinity GLP-1RA on kidney tubular dysregulation in diabetes and the possible mechanisms involved, with focus on ferroptosis and adenosine 5'-monophosphate-activated protein kinase (AMPK)-mitochondrial lipid metabolism pathway. Our data revealed that exendin-4 treatment markedly improved kidney structure and function by reducing iron overload, oxidative stress, and ACSL4-driven lipid peroxidation taken place in diabetic kidney tubules, along with reduced GPX4 expression and GSH content. AMPK signaling was identified as the downstream target of exendin-4, and enhancement of AMPK triggered the transmit of its downstream signal to activate fatty acid oxidation in mitochondria and suppress lipid synthesis and glycolysis, and ultimately alleviated toxic lipid accumulation and ferroptosis. Further study suggested that exendin-4 was taken up by tubular cells via macropinocytosis. The protective effect of exendin-4 on tubular ferroptosis was abolished by macropinocytosis blockade. Taken together, present work demonstrated the beneficial effects of GLP-1RA treatment on kidney tubular protection in diabetes by suppressing ferroptosis through enhancing AMPK-fatty acid metabolic signaling via macropinocytosis.

Impact of short and long exposure to cafeteria diet on food intake and white adipose tissue lipolysis mediated by glucagon-like peptide 1 receptor.

Introduction: The modern food environment facilitates excessive calorie intake, a major driver of obesity. Glucagon-like peptide 1 (GLP1) is a neuroendocrine peptide that has been the basis for developing new pharmacotherapies against obesity. The GLP1 receptor (GLP1R) is expressed in central and peripheral tissues, and activation of GLP1R reduces food intake, increases the expression of thermogenic proteins in brown adipose tissue (BAT), and enhances lipolysis in white adipose tissue (WAT). Obesity decreases the efficiency of GLP1R agonists in reducing food intake and body weight. Still, whether palatable food intake before or during the early development of obesity reduces the effects of GLP1R agonists on food intake and adipose tissue metabolism remains undetermined. Further, whether GLP1R expressed in WAT contributes to these effects is unclear. Methods: Food intake, expression of thermogenic BAT proteins, and WAT lipolysis were measured after central or peripheral administration of Exendin-4 (EX4), a GLP1R agonist, to mice under intermittent-short exposure to CAF diet (3 h/d for 8 days) or a longer-continuous exposure to CAF diet (24 h/d for 15 days). Ex-vivo lipolysis was measured after EX4 exposure to WAT samples from mice fed CAF or control diet for 12 weeks. . Results: During intermittent-short exposure to CAF diet (3 h/d for 8 days), third ventricle injection (ICV) and intra-peritoneal administration of EX4 reduced palatable food intake. Yet, during a longer-continuous exposure to CAF diet (24 h/d for 15 days), only ICV EX4 administration reduced food intake and body weight. However, this exposure to CAF diet blocked the increase in uncoupling protein 1 (UCP1) caused by ICV EX4 administration in mice fed control diet. Finally, GLP1R expression in WAT was minimal, and EX4 failed to increase lipolysis ex-vivo in WAT tissue samples from mice fed CAF or control diet for 12 weeks. . Discussion: Exposure to a CAF diet during the early stages of obesity reduces the effects of peripheral and central GLP1R agonists, and WAT does not express a functional GLP1 receptor. These data support that exposure to the obesogenic food environment, without the development or manifestation of obesity, can alter the response to GLP1R agonists. .

A Research Update on Exendin-4 as a Novel Molecule Against Parkinson's Disease.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder, and its consequences severely influence the quality of a patient's life and mobility. PD is characterized by bradykinesias with tremors and/or rigidity. Pathophysiologically, PD is associated with the gradual degeneration of dopaminergic neurons in the substantia nigra pars compacta of the midbrain, neuroinflammation, increased accumulation of the alpha (α)-synuclein, overburden of oxidative stress, and mitochondrial dysfunction. To date, there are no effective therapies with underlying shreds of evidence that alters the progression of PD. Exendin-4, a glucagon-like peptide 1 (GLP-1) receptor agonist, has gained attention for its tremendous neuroprotective potential against numerous neurodegenerative disorders, including PD. Further, several pieces of research evidence have suggested the beneficial role of Exendin-4 in PD-like experimental models. The present review article highlights the preclinical and clinical evidence of the therapeutic benefits of Exendin-4 against PD. Exendin-4 reverses the PD-like symptoms in experimental animals by dramatically minimizing the loss of dopaminergic neuronal and accumulation of α-synuclein in the PD-like brain. Further, it also reduces the mitochondrial toxicity and expression of pro-inflammatory mediators such as tumor necrosis factor (TNF)-α and interleukin (IL)-1ß. These observations designate that Exendin-4 is a multifactorial compound that could be considered a safe, effective, and new ingredient for developing clinically useful pharmacotherapy for managing PD-like manifestations.

The effect of human serum albumin glycation on the binding of antidiabetic agent-exenatide; the multispectroscopic studies. Part II.

The glycated human serum albumin (gHSA) interaction with antidiabetic agent - exenatide (exe) was investigated by spectroscopic techniques. The fluorescence spectroscopy showed an association between exe and protein as 4.57 × 104 M-1 (290 K), 3.33 × 104 M-1 (300K) and 2.54 × 104 M-1 (310K) and the number of binding sites were 1.15, 1.09, and 1.02, respectively. The binding process occurred spontaneously and the electrostatic bonds play a predominant role in the gHSA-exe interaction. The affinity drug to gHSA was studied in the presence of metal ions (Ca2+, Cr3+, Zn2+), which are often given as a supplement to the diet. Our study indicates that metal ions have an effect on the increase of the affinity of exenatide to glycated albumin and it is the result of the metal ion-exenatide-albumin interaction. Based on CD spectroscopy it is evident that exenatide has nonsignificant effect on the protein secondary structure, but the changes are visible in the presence of metal ions. Zeta potential measurements indicate instability of the system at lower concentration of the ligand - the effect is desired in point of view of pharmacology.

Defining the Intravital Renal Disposition of Fluorescence-Quenched Exenatide.

Despite the understanding that renal clearance is pivotal for driving the pharmacokinetics of numerous therapeutic proteins and peptides, the specific processes that occur following glomerular filtration remain poorly defined. For instance, sites of catabolism within the proximal tubule can occur at the brush border, within lysosomes following endocytosis, or even within the tubule lumen itself. The objective of the current study was to address these limitations and develop methodology to study the kidney disposition of a model therapeutic protein. Exenatide is a peptide used to treat type 2 diabetes mellitus. Glomerular filtration and ensuing renal catabolism have been shown to be its principal clearance pathway. Here, we designed and validated a Förster resonance energy transfer-quenched exenatide derivative to provide critical information on the renal handling of exenatide. A combination of in vitro techniques was used to confirm substantial fluorescence quenching of intact peptide that was released upon proteolytic cleavage. This evaluation was then followed by an assessment of the in vivo disposition of quenched exenatide directly within kidneys of living rats via intravital two-photon microscopy. Live imaging demonstrated rapid glomerular filtration and identified exenatide metabolism occurred within the subapical regions of the proximal tubule epithelia, with subsequent intracellular trafficking of cleaved fragments. These results provide a novel examination into the real-time, intravital disposition of a protein therapeutic within the kidney and offer a platform to build upon for future work.

Effects of antidiabetics and exercise therapy on suppressors of cytokine signaling-1, suppressors of cytokine signaling-3, and insulin receptor substrate-1 molecules in diabetes and obesity.

OBJECTIVE: Pathological destruction of insulin signaling molecules such as insulin receptor substrate, especially due to the increase in suppressors of cytokine signaling molecules, has been demonstrated in experimental diabetes. The contribution of suppressors of cytokine signaling proteins to the development of insulin resistance and the effects of antidiabetic drugs and exercise on suppressors of cytokine signaling proteins are not clearly known. METHODS: A total of 48 Wistar albino adult male rats were divided into six groups: control group, obese group with diabetes, obese diabetic rats treated with metformin, obese diabetic rats treated with pioglitazone, obese diabetic rats treated with exenatide, and obese diabetic rats with applied exercise program. Immunohistochemical staining was performed in both the liver and adipose tissue. RESULTS: There was a statistically significant decrease in suppressors of cytokine signaling-1, a decrease in suppressors of cytokine signaling-3, an increase in insulin receptor substrate-1, and a decrease in immunohistochemical staining in the obese group treated with metformin and exenatide compared to the obese group without treatment in the liver tissue (p<0.05). A statistically significant decrease in immunohistochemical staining of suppressors of cytokine signaling-1 and suppressors of cytokine signaling-3 was found in the obese group receiving exercise therapy compared to the obese group without treatment in visceral adipose tissue (p<0.05). Likewise, no significant immunohistochemistry staining was seen in diabetic obese groups. CONCLUSION: Metformin or exenatide treatment could prevent the degradation of insulin receptor substrate-1 protein by reducing the effect of suppressors of cytokine signaling-1 and suppressors of cytokine signaling-3 proteins, especially in the liver tissue. In addition, exercise can play a role as a complementary therapy by reducing suppressors of cytokine signaling-1 and suppressors of cytokine signaling-3 proteins in visceral adipose tissue.

Conjugation to a cell-penetrating peptide drives the tumour accumulation of the GLP1R antagonist exendin(9-39).

PURPOSE: Exendin, an analogue of the glucagon-like peptide 1 (GLP1), is an excellent tracer for molecular imaging of pancreatic beta cells and beta cell-derived tumours. The commonly used form, exendin-4, activates the GLP1 receptor and causes internalisation of the peptide-receptor complex. As a consequence, injection of exendin-4 can lead to adverse effects such as nausea, vomiting and hypoglycaemia and thus requires close monitoring during application. By comparison, the antagonist exendin(9-39) does not activate the receptor, but its lack of internalisation has precluded its use as a tracer. Improving the cellular uptake of exendin(9-39) could turn it into a useful alternative tracer with less side-effects than exendin-4. METHODS: We conjugated exendin-4 and exendin(9-39) to the well-known cell-penetrating peptide (CPP) penetratin. We evaluated cell binding and internalisation of the radiolabelled peptides in vitro and their biodistribution in vivo. RESULTS: Exendin-4 showed internalisation irrespective of the presence of the CPP, whereas for exendin(9-39) only the penetratin conjugate internalised. Conjugation to the CPP also enhanced the in vivo tumour uptake and retention of exendin(9-39). CONCLUSION: We demonstrate that penetratin robustly improves internalisation and tumour retention of exendin(9-39), opening new avenues for antagonist-based in vivo imaging of GLP1R.

Glucagon-like peptide-1 receptors in nucleus accumbens, ventral hippocampus, and lateral septum reduce alcohol reinforcement in mice.

Glucagon-like peptide 1 (GLP-1) receptor agonists can decrease alcohol intake by central mechanisms that are still poorly understood. The lateral septum (LS) and the ventral/caudal part of the hippocampus are enriched in GLP-1 receptors, and activity in these regions was shown to modulate reward-related behaviors. Using microinfusions of the GLP-1 receptor agonist exendin-4 in mice trained to self-administer oral alcohol in an operant assay, we tested whether pharmacological stimulation of GLP-1 receptors in hippocampus and LS decrease alcohol self-administration. We report that infusion of exendin-4 in the ventral hippocampus or LS was sufficient to reduce alcohol self-administration with as large effect sizes as we previously reported with systemic exendin-4 administration. Infusion of exendin-4 into the nucleus accumbens also reduced alcohol self-administration, as anticipated based on earlier reports, while infusion of exendin-4 into the caudate-putamen (dorsal striatum) had little effect, consistent with lack of GLP-1 receptor expression in this region. The distribution of exendin-4 after infusion into the LS or caudate putamen was visualized using a fluorescently labeled ligand. These findings add to our understanding of the circuit-level mechanisms underlying the ability of GLP-1 receptor agonists to reduce alcohol self-administration. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Exenatide improves hepatocyte insulin resistance induced by different regional adipose tissue.

Obesity is resulted from energy surplus and is characterized by abnormal adipose tissue accumulation and/or distribution. Adipokines secreted by different regional adipose tissue can induce changes in key proteins of the insulin signaling pathway in hepatocytes and result in impaired hepatic glucose metabolism. This study aimed to investigate whether exenatide affects key proteins of IRS2/PI3K/Akt2 signaling pathway in hepatocytes altered by the different regional fat depots. Six non-obese patients without endocrine diseases were selected as the research subjects. Their subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT)were co-cultured with HepG2 cells in the transwell chamber. In the presence or absence of exenatide, adipokines content in the supernatant of each experimental group was detected by ELISA. In addition, HepG2 cells in each co-culture group with and without insulin were collected, and the expression of key proteins IRS2, p-IRS2(S731), PI3K-p85, Akt2, and p-Akt2(S473) was detected by western blotting (WB). The results showed that the adipokines IL-8, MCP-1, VEGF, and sTNFR2 in the supernatant of HepG2 cells induced by different regional adipose tissue were significantly higher than those in the HepG2 group, and VAT released more adipokines than SAT. Furthermore, these adipokines were significantly inhibited by exenatide. Importantly, the different regional fat depot affects the IRS2/PI3K/Akt2 insulin signaling pathway of hepatocytes. Exenatide can up-regulate the expression of hepatocyte proteins IRS2, PI3K-p85, p-Akt2(S731) inhibited by adipose tissue, and down-regulate the expression of hepatocyte proteins p-IRS2(S731) promoted by adipose tissue. The effect of VAT on the expression of these key proteins in hepatocytes is more significant than that of SAT. But there was no statistical difference in the expression of Akt2 protein among each experimental group, suggesting that exenatide has no influence on the expression of Akt2 protein in hepatocytes. In conclusion, exenatide may improve hepatic insulin resistance (IR) by inhibiting adipokines and regulating the expression of key proteins in the IRS2/PI3K/Akt2 pathway.

Glucagon-like peptide-1 receptor differentially controls mossy cell activity across the dentate gyrus longitudinal axis.

Understanding the role of dentate gyrus (DG) mossy cells (MCs) in learning and memory has rapidly evolved due to increasingly precise methods for targeting MCs and for in vivo recording and activity manipulation in rodents. These studies have shown MCs are highly active in vivo, strongly remap to contextual manipulation, and that their inhibition or hyperactivation impairs pattern separation and location or context discrimination. Less well understood is how MC activity is modulated by neurohormonal mechanisms, which might differentially control the participation of MCs in cognitive functions during discrete states, such as hunger or satiety. In this study, we demonstrate that glucagon-like peptide-1 (GLP-1), a neuropeptide produced in the gut and the brain that regulates food consumption and hippocampal-dependent mnemonic function, might regulate MC function through expression of its receptor, GLP-1R. RNA-seq demonstrated that most, though not all, Glp1r in hippocampal principal neurons is expressed in MCs, and in situ hybridization revealed strong expression of Glp1r in hilar neurons. Glp1r-ires-Cre mice crossed with Ai14D reporter mice followed by co-labeling for the MC marker GluR2/3 revealed that almost all MCs in the ventral DG expressed Glp1r and that almost all Glp1r-expressing hilar neurons were MCs. However, only ~60% of dorsal DG MCs expressed Glp1r, and Glp1r was also expressed in small hilar neurons that were not MCs. Consistent with this expression pattern, peripheral administration of the GLP-1R agonist exendin-4 (5 µg/kg) increased cFos expression in ventral but not dorsal DG hilar neurons. Finally, whole-cell patch-clamp recordings from ventral MCs showed that bath application of exendin-4 (200 nM) depolarized MCs and increased action potential firing. Taken together, this study adds to known MC activity modulators a neurohormonal mechanism that may preferentially affect ventral DG physiology and may potentially be targetable by several GLP-1R pharmacotherapies already in clinical use.

Structure-Activity Relationships and Pharmacokinetics of 111In-Labeled Glucagon-like Peptide-1 Receptor-Targeting Exendin-4 Derivatives Conjugated with Albumin Binder Moieties.

Insulinomas are neuroendocrine tumors that are derived from pancreatic ß-cells, and they often overexpress the glucagon-like peptide-1 receptor (GLP-1R). Radiolabeled exendin-4 derivatives have been used to noninvasively detect the GLP-1R during the diagnosis and preoperative localization of insulinomas; however, their marked renal accumulation can hinder the imaging of pancreatic tail lesions. In this study, we designed and synthesized 111In-labeled exendin-4 derivatives that possessed 4-(4-substituted phenyl)-moieties as albumin binder (ALB) moieties ([111In]In-E4DA2-4), and studied their structure-activity relationships and pharmacokinetics (as well as those of [111In]In-E4DA1, which we previously reported) to determine their usefulness as radioligands for GLP-1R imaging. 111In-labeling was performed by reacting maleimide precursors with [111In]InCl3 in 2-(N-morpholino)ethanesulfonic acid buffer, and then, the products were conjugated with exendin-4-Cys40. A saturation binding assay using GLP-1R-expressing INS-1 cells was carried out to evaluate the in vitro affinity of the radioligands for the cells. In addition, the affinity of the 111In-labeled derivatives for human serum albumin (HSA) was evaluated in an HSA-binding assay. Furthermore, an in vivo biodistribution study and single-photon emission computed tomography (SPECT) imaging were performed using INS-1 tumor-bearing mice. [111In]In-E4DA1-4 were prepared at radiochemical yields of 6-17%. In the saturation binding assay, [111In]In-E4DA1-4 showed a similar affinity for the INS-1 cells, indicating that the kind of ALB moiety used had no effect on the affinity of the exendin-4 derivatives for the cells. In the HSA-binding assay, [111In]In-E4DA1-4 all bound to HSA. In the biodistribution assay, [111In]In-E4DA1-4 exhibited marked tumor accumulation and retention. In addition, they showed lower renal accumulation than previously reported exendin-4-based radioligands without ALB moieties. The pharmacokinetics of the 111In-labeled exendin-4 derivatives varied markedly according to the kind of ALB moiety used. In particular, [111In]In-E4DA2, which contained a 4-(4-bromophenyl)butyric acid derivative as an ALB moiety, showed the highest tumor accumulation. SPECT imaging with [111In]In-E4DA2 clearly visualized INS-1 tumors with no marked accumulation in normal organs. These results provide important information that will aid the design of novel exendin-4-based radioligands targeting the GLP-1R.

Exenatide enhanced the antitumor efficacy on PD-1 blockade by the attenuation of neutrophil extracellular traps.

Neutrophil extracellular traps (NETs) are extracellular webs of DNA, histones, and granular contents, such as myeloperoxidase (MPO) and elastase, which are released by neutrophils. Reactive oxygen species (ROS) are involved in NETs formation that promote tumor progress. Exenatide could downregulate ROS production in some cell types. However, it is unknown whether Exenatide could influence tumor progress through NETs. Here, we constructed the LLC-based lung cancer and MC38-based colon cancer models and found that Exenatide treatment decreased tumor infiltrated NETs and peripheral MPO-DNA complex and elastase. In addition, the in vitro study showed that Exenatide decreased NETs formation and release. Furthermore, flow cytometry analysis showed that Exenatide treatment reduced ROS production in tumor infiltrated and in vitro neutrophils. However, the ROS inhibitor DPI counteracted the decease of tumor infiltrated and in vitro NETs formation and release by Exenatide. Functionally, the Exenatide/αPD-1 combination therapy was superior to single therapy in restricting tumor growth. Removement of NETs by DNase I weaken the enhancement of αPD-1 treatment by Exenatide. The enriched tumor infiltrated, spleen and lymph node CD8+ T cells from combination therapy group secreted higher concentration of IFN-γ than single treatment. In addition, Exenatide exhibited no direct influence on IFN-γ secretion while purified NETs decreased IFN-γ secretion by CD8+ T cells. The rechallenge study showed that the combination therapy activated long-term tumor rejection. In summary, our findings suggested that Exenatide might be a promising therapeutic candidate for enhancing PD-1 blockade in tumor treatment.

Signaling Mechanism for Modulation by GLP-1 and Exendin-4 of GABA Receptors on Rat Retinal Ganglion Cells.

Glucagon-like peptide-1 (GLP-1) is expressed in retinal neurons, but its role in the retina is largely unknown. Here, we demonstrated that GLP-1 or the GLP-1 receptor (GLP-1R; a G protein-coupled receptor) agonist exendin-4 suppressed γ-aminobutyric acid receptor (GABAR)-mediated currents through GLP-1Rs in isolated rat retinal ganglion cells (GCs). Pre-incubation with the stimulatory G protein (Gs) inhibitor NF 449 abolished the exendin-4 effect. The exendin-4-induced suppression was mimicked by perfusion with 8-Br-cAMP (a cAMP analog), but was eliminated by the protein kinase A (PKA) inhibitor Rp-cAMP/KT-5720. The exendin-4 effect was accompanied by an increase in [Ca2+]i of GCs through the IP3-sensitive pathway and was blocked in Ca2+-free solution. Furthermore, when the activity of calmodulin (CaM) and CaM-dependent protein kinase II (CaMKII) was inhibited, the exendin-4 effect was eliminated. Consistent with this, exendin-4 suppressed GABAR-mediated light-evoked inhibitory postsynaptic currents in GCs in rat retinal slices. These results suggest that exendin-4-induced suppression may be mediated by a distinct Gs/cAMP-PKA/IP3/Ca2+/CaM/CaMKII signaling pathway, following the activation of GLP-1Rs.

Activation of glucagon-like peptide-1 receptor in microglia attenuates neuroinflammation-induced glial scarring via rescuing Arf and Rho GAP adapter protein 3 expressions after nerve injury.

Rationale: The neuroinflammation is necessary for glial group initiation and clearance of damaged cell debris after nerve injury. However, the proinflammatory polarization of excessive microglia amplifies secondary injury via enhancing cross-talk with astrocytes and exacerbating neurological destruction after spinal cord injury (SCI). The glucagon-like peptide-1 receptor (GLP-1R) agonist has been previously shown to have a neuroprotective effect in neurodegeneration, whereas its potency in microglial inflammation after SCI is still unknown. Methods: The effect and mechanism of GLP-1R activation by exendin-4 (Ex-4) were investigated in in vitro cultured glial groups and in vivo in SCI mice. Alterations in the gene expression after GLP-1R activation in inflammatory microglia were measured using mRNA sequencing. The microglial polarization, neuroinflammatory level, and astrocyte reaction were detected by using western blotting, flow cytometry, and immunofluorescence. The recoveries of neurological histology and function were also observed using imaging and ethological examinations. Results: GLP-1R activation attenuated microglia-induced neuroinflammation by reversing M1 subtypes to M2 subtypes in vitro and in vivo. In addition, activation of GLP-1R in microglia blocked production of reactive astrocytes. We also found less neuroinflammation, reactive astrocytes, corrected myelin integrity, ameliorated histology, and improved locomotor function in SCI mice treated with Ex-4. Mechanistically, we found that Ex-4 rescued the RNA expression of Arf and Rho GAP adapter protein 3 (ARAP3). Knockdown of ARAP3 in microglia reversed activation of RhoA and the pharmacological effect of Ex-4 on anti-inflammation in vitro. Conclusion: Ex-4 exhibited a previously unidentified role in reducing reactive astrocyte activation by mediation of the PI3K/ARAP3/RhoA signaling pathway, by neuroinflammation targeting microglia, and exerted a neuroprotective effect post-SCI, implying that activation of GLP-1R in microglia was a therapeutical option for treatment of neurological injury.

Exendin-4 enhances osteogenic differentiation of adipose tissue mesenchymal stem cells through the receptor activator of nuclear factor-kappa B and osteoprotegerin signaling pathway.

The capability of mesenchymal stem cells (MSCs) to repair bone damage and defects has long been investigated. The receptor activator of nuclear factor-kappa B (RANK), its ligand (RANKL) and the decoy receptor osteoprotegerin (OPG) axis is crucial to keep the equilibrium between osteoblastic and osteoclastic activity. Exendin-4 utilization increased bone formation and enhanced bone integrity. This study aimed to investigate the mentioned axis and determine the effect of exendin-4 upon adipose mesenchymal stem cells (Ad-MSCs) osteogenic differentiation. Ad-MSCs were isolated from rat epididymal fat, followed by characterization and then differentiation into osteocytes both in the presence or absence of exendin-4. Osteogenic differentiation was evaluated by alizarin red staining and the expression of osteogenic markers; using reverse transcriptase-quantitative polymerase chain reaction, western blotting and enzyme-linked immunoassay. MSCs derived from rat epididymal fat were isolated and characterized, along with their differentiation into osteocytes. The differentiated cells were alizarin red-stained, showing increased staining intensity upon addition of exendin-4. Moreover, the addition of exendin-4 elevated the messenger RNA expression levels of osteogenic markers; runt-related transcription factor-2 (RUNX-2), osteocalcin, and forkhead box protein O-1 while reducing the expression of the adipogenic marker peroxisome-proliferator-activated receptor-gamma. Exendin-4 addition elevated OPG levels in the supernatant of osteogenic differentiated cells. Moreover, exendin-4 elevated the protein levels of glucagon-like peptide-1 receptor and RUNX-2, while decreasing both RANK and RANKL. In conclusion, osteogenic differentiation of Ad-MSCs is associated with increased osteoblastic rather than osteoclastic activity. The findings of this study suggest that exendin-4 can enhance Ad-MSCs osteogenic differentiation partially through the RANK/RANKL/OPG axis.

Exendin-4 and eldecalcitol synergistically promote osteogenic differentiation of bone marrow mesenchymal stem cells through M2 macrophages polarization via PI3K/AKT pathway.

BACKGROUND: The incidence of diabetic osteoporosis is increasing. This article evaluates the effect of combination treatment with the hypoglycemic drug exendin-4 (Ex-4) and the vitamin D analog eldecalcitol (ED-71) on improving diabetic osteoporosis and explores the relevant mechanism of action. METHOD: Micro-CT, HE staining, immunohistochemistry, qPCR and ELISA were used to evaluate the impact of Ex-4 and ED-71 on bone formation and macrophage polarization in a mouse model of diabetic osteoporosis in vivo. Immunofluorescence, flow cytometry and qPCR were used to characterize the polarization type of macrophages treated with Ex-4 and ED-71 in vitro. A co-culture system of BMSCs and macrophages was established. Subsequently, crystal violet staining, alkaline phosphatase staining and alizarin red staining were used to evaluate the migration and osteogenesis differentiation of BMSCs. RESULTS: Ex-4 combined with ED-71 significantly reduced blood glucose levels and enhanced bone formation in mice with diabetic osteoporosis. In addition, Ex-4 synergized with ED-71 to induce the polarization of macrophages into M2 through the PI3K/AKT pathway. Macrophages treated with the combination of Ex-4 and ED-71 can significantly induce the osteogenic differentiation of BMSCs. CONCLUSION: Ex-4 synergized with ED-71 to reduce blood glucose levels significantly. And this combination therapy can synergistically induce osteogenic differentiation of BMSCs by promoting M2 macrophages polarization, thereby improving diabetic osteoporosis. Therefore, the combination of Ex-4 and ED-71 may be a new strategy for the treatment of diabetic osteoporosis.

The effects of metformin, pioglitazone, exenatide and exercise on fatty liver in obese diabetic rats: the role of IRS-1 and SOCS-3 molecules.

BACKGROUND: Obesity-induced inflammation mechanism is seen as a mechanism that may be the cause of insulin resistance and non-alcoholic fatty liver disease (NAFLD). Pathological destruction of insulin signaling molecules such as insulin receptor substrate proteins (IRS), especially due to the increase of cytokine signal suppressors (SOCS), has been demonstrated in experimental diabetes. The aim of this study was to determine the effects of metformin, pioglitazone, exenatide and exercise treatments used in type 2 diabetes on fatty liver and the role of Irs-1 and Socs3 molecules in this process in obese diabetic rats. METHODS: The study was conducted on 48 Wistar albino adult male rats weighing 180-220 g and randomly divided into 6 groups. The obese rat model with fatty liver was formed with a 60% fat diet for 4 weeks. Afterwards, drug treatment with metformin (Ob + D + M), pioglitazone (Ob + D + P), exenatide (Ob + D + ExA)) or exercise (Ob + D + ExE) was applied for 4 weeks to these obese groups, in which diabetes was induced by streptozocin (STZ). At the end of the experimental protocol, liver tissue samples were taken from all rat groups and histopathological and genetic analyses were performed. RESULTS: The mean steatosis degrees of the Ob + D + ExA and Ob + D + ExE groups were statistically significantly decreased compared to the obese diabetic group (p < 0.001). The group with the lowest mean steatosis grade was the Ob + D + ExE. Decrease in SOCS-3 expression was significant in Ob + D + M and Ob + D + P groups than other groups (p < 0.05). Mean staining intensities of Ob + D + Ex group, Ob + D + ExE group and Ob + D + P group according to IRS-1 expression statistically significantly increased compared to obese diabetic group (p < 0.05). Average staining intensity of Ob + D + ExE group according to IRS-1 expression was significant than other groups. CONCLUSION: Exercise and exenatide treatments seemed to be the prominent treatment methods by showing a statistically significant effect in decreasing the degree of steatosis, decreasing the Socs3 expression level and increasing the Irs-1 expression level.

Dynamics of GLP-1R peptide agonist engagement are correlated with kinetics of G protein activation.

The glucagon-like peptide-1 receptor (GLP-1R) has broad physiological roles and is a validated target for treatment of metabolic disorders. Despite recent advances in GLP-1R structure elucidation, detailed mechanistic understanding of how different peptides generate profound differences in G protein-mediated signalling is still lacking. Here we combine cryo-electron microscopy, molecular dynamics simulations, receptor mutagenesis and pharmacological assays, to interrogate the mechanism and consequences of GLP-1R binding to four peptide agonists; glucagon-like peptide-1, oxyntomodulin, exendin-4 and exendin-P5. These data reveal that distinctions in peptide N-terminal interactions and dynamics with the GLP-1R transmembrane domain are reciprocally associated with differences in the allosteric coupling to G proteins. In particular, transient interactions with residues at the base of the binding cavity correlate with enhanced kinetics for G protein activation, providing a rationale for differences in G protein-mediated signalling efficacy from distinct agonists.

Exenatide prevents statin-related LDL receptor increase and improves insulin secretion in pancreatic beta cells (1.1E7) in a protein kinase A-dependent manner.

Statins are primary drugs in the treatment of hyperlipidemias. This group of drugs is known for its beneficial pleiotropic effects (e.g., reduction of inflammatory state). However, a growing body of evidence suggests its diabetogenic properties. The culpable mechanism is not completely understood and might be related to the damage to pancreatic beta cells. Therefore, we conceived an in vitro study to explore the impact of atorvastatin on pancreatic islet beta cells line (1.1.E7). We evaluated the influence on viability, insulin, low-density lipoprotein (LDL) receptor, and proprotein convertase subtilisin/kexin type 9 (PCSK9) expression. A significant drop in mRNA for proinsulin and insulin expression was noted. Concurrently, a rise in LDL receptor at the protein level in cells exposed to atorvastatin was noted. Further experiments have shown that exenatide - belonging to glucagon-like peptide 1 (GLP-1) analogs that are used in a treatment of diabetes and known for its weight reducing properties - can alleviate the observed alterations. In this case, the mechanism of action of exenatide was dependent on a protein kinase A pathway. In conclusion, our results support the hypothesis that statin may have diabetogenic properties, which according to our study is related to reduced insulin expression. The concomitant use of GLP-1 receptor agonist seemed to successfully revert insulin expression.

Exendin-4 Pretreatment Attenuates Kainic Acid-Induced Hippocampal Neuronal Death.

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.