Functional maturation and longitudinal imaging of intraportal neonatal porcine islet grafts in genetically diabetic pigs.

Allogeneic intraportal islet transplantation (ITx) has become an established treatment for patients with poorly controlled type 1 diabetes. However, the loss of viable beta-cell mass after transplantation remains a major challenge. Therefore, noninvasive imaging methods for long-term monitoring of the transplant fate are required. In this study, [68Ga]Ga-DOTA-exendin-4 positron emission tomography/computed tomography (PET/CT) was used for repeated monitoring of allogeneic neonatal porcine islets (NPI) after intraportal transplantation into immunosuppressed genetically diabetic pigs. NPI transplantation (3320-15,000 islet equivalents per kg body weight) led to a reduced need for exogenous insulin therapy and finally normalization of blood glucose levels in 3 out of 4 animals after 5 to 10 weeks. Longitudinal PET/CT measurements revealed a significant increase in standard uptake values in graft-bearing livers. Histologic analysis confirmed the presence of well-engrafted, mature islet clusters in the transplanted livers. Our study presents a novel large animal model for allogeneic intraportal ITx. A relatively small dose of NPIs was sufficient to normalize blood glucose levels in a clinically relevant diabetic pig model. [68Ga]Ga-DOTA-exendin-4 PET/CT proved to be efficacious for longitudinal monitoring of islet transplants. Thus, it could play a crucial role in optimizing ITx as a curative therapy for type 1 diabetes.

Targeting autophagy to counteract neuroinflammation: A novel antidepressant strategy.

Depression is a common disease that affects physical and mental health and imposes a considerable burden on afflicted individuals and their families worldwide. Depression is associated with a high rate of disability and suicide. It causes a severe decline in productivity and quality of life. Unfortunately, the pathophysiological mechanisms underlying depression have not been fully elucidated, and the risk of its treatment is still presented. Studies have shown that the expression of autophagic markers in the brain and peripheral inflammatory mediators are dysregulated in depression. Autophagy-related genes regulate the level of autophagy and change the inflammatory response in depression. Depression is related to several aspects of immunity. The regulation of the immune system and inflammation by autophagy may lead to the development or deterioration of mental disorders. This review highlights the role of autophagy and neuroinflammation in the pathophysiology of depression, sumaries the autophagy-targeting small moleculars, and discusses a novel therapeutic strategy based on anti-inflammatory mechanisms that target autophagy to treat the disease.

Exendin-4 blockade of T1R2/T1R3 activation improves Pseudomonas aeruginosa-related pneumonia in an animal model of chemically induced diabetes.

OBJECTIVE: Poorly controlled diabetes frequently exacerbates lung infection, thereby complicating treatment strategies. Recent studies have shown that exendin-4 exhibits not only hypoglycemic but also anti-inflammatory properties. This study aimed to explore the role of exendin-4 in lung infection with diabetes, as well as its association with NOD1/NF-κB and the T1R2/T1R3 sweet taste receptor. METHODS: 16HBE human bronchial epithelial cells cultured with 20 mM glucose were stimulated with lipopolysaccharide (LPS) isolated from Pseudomonas aeruginosa (PA). Furthermore, Sprague‒Dawley rats were fed a high-fat diet, followed by intraperitoneal injection of streptozotocin and intratracheal instillation of PA. The levels of TNF-α, IL-1ß and IL-6 were evaluated using ELISAs and RT‒qPCR. The expression of T1R2, T1R3, NOD1 and NF-κB p65 was assayed using western blotting and immunofluorescence staining. Pathological changes in the lungs of the rats were observed using hematoxylin and eosin (H&E) staining. RESULTS: At the same dose of LPS, the 20 mM glucose group produced more proinflammatory cytokines (TNF-α, IL-1ß and IL-6) and had higher levels of T1R2, T1R3, NOD1 and NF-κB p65 than the normal control group (with 5.6 mM glucose). However, preintervention with exendin-4 significantly reduced the levels of the aforementioned proinflammatory cytokines and signaling molecules. Similarly, diabetic rats infected with PA exhibited increased levels of proinflammatory cytokines in their lungs and increased expression of T1R2, T1R3, NOD1 and NF-κB p65, and these effects were reversed by exendin-4. CONCLUSIONS: Diabetic hyperglycemia can exacerbate inflammation during lung infection, promote the increase in NOD1/NF-κB, and promote T1R2/T1R3. Exendin-4 can ameliorate PA-related pneumonia with diabetes and overexpression of NOD1/NF-κB. Additionally, exendin-4 suppresses T1R2/T1R3, potentially through its hypoglycemic effect or through a direct mechanism. The correlation between heightened expression of T1R2/T1R3 and an intensified inflammatory response in lung infection with diabetes requires further investigation.

Exendin-4 alleviates myocardial ischemia reperfusion injury by enhancing autophagy through promoting nuclear translocation of TFEB.

Ischemia-reperfusion (I/R) injury (IRI) is a common clinical consequence of myocardial infarction. Exendin-4 is a glucagon-like peptide-1 (GLP-1) analog that has been demonstrated to alleviate myocardial IRI. Autophagy, a lysosomal pathway balancing cell survival and cell death, is engaged in myocardial IRI. However, whether exendin-4 exerts a protective effect on myocardial IRI by modulating autophagy remains elusive. Herein, we investigated the effect of exendin-4 on autophagic flux and explored the underlying molecular mechanisms. Our data revealed that the autophagic flux was blocked in the human ventricular cardiomyocyte cell lines (AC16) subjected to oxygen glucose deprivation/reoxygenation (OGD/R) in vitro. Exendin-4 pre-treatment markedly restored the blocked autophagic flux induced by OGD/R through promoting nuclear translocation of TFEB and transcription of genes involving autophagy initiation, the effect of which was reversed by TFEB knockdown. The restoration of autophagic flux contributed to multiple beneficial effects of exendin-4 in cardiomyocytes, including reduction of oxidative stress, preservation of mitochondrial network as well as inhibition of cytochrome c leakage from mitochondrial permeability transition pore (MPTP) and the resulting apoptosis. Moreover, the administration of exendin-4 reduced infarct size and preserved cardiac function through its anti-apoptosis and antioxidative effects in vivo. These results shed some light on understanding the novel mechanism of exendin-4 as a protective agent against myocardial IRI.

[18F]FB(ePEG12)12-exendin-4 noninvasive imaging of insulinoma negative for insulin immunostaining on specimen from endoscopic ultrasonography-guided fine needle aspiration: a case report with review of literature.

Insulinomas are the most common functional pancreatic neuroendocrine neoplasm; when treatment is delayed, they induce hyperinsulinemic hypoglycemia, which is life-threatening. As surgical resection is the only curative treatment for insulinoma, preoperative localization is crucial; however, localization based on conventional imaging modalities such as computed tomography (CT) and magnetic resonance imaging is often inconclusive. Somatostatin receptor-targeted imaging is another option for detecting pancreatic neuroendocrine neoplasms but has low sensitivity and is not specific for insulinoma. The clinical application of other localizing approaches such as selective arterial calcium stimulation and endoscopic ultrasonography-guided fine needle aspiration (EUS-FNA) is limited by their being invasive and/or technically complex. Moreover, an EUS-FNA specimen of an insulinoma may be negative on insulin immunostaining. Thus, a noninvasive and clinically practical insulinoma-specific diagnostic tool to discriminate insulinomas with high accuracy is anticipated. Glucagon-like peptide-1 receptor (GLP-1R)-targeted imaging has emerged in the effort to fulfill this need. We recently developed the novel fluorine-18-labeled exendin-4-based probe conjugated with polyethylene glycol, [18F]FB(ePEG12)12-exendin-4 (18F-exendin-4) for positron emission tomography (PET) imaging and reported its clinical benefit in a case of insulinoma in the pancreatic tail. We report here a case of insulinoma in the pancreatic head in which an EUS-FNA specimen was negative on insulin immunostaining while precise preoperative localization and conclusive evidence for curative enucleation was provided by 18F-exendin-4 PET/CT (Japan Registry of Clinical Trials; jRCTs051200156).

GLP-1 Receptor Agonists: A New Treatment in Parkinson's Disease.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Recent data highlight similarities between neurodegenerative diseases, including PD and type 2 diabetes mellitus (T2DM), suggesting a crucial interplay between the gut-brain axis. Glucagon-like peptide-1 receptor (GLP-1R) agonists, known for their use in T2DM treatment, are currently extensively studied as novel PD modifying agents. For this narrative review article, we searched PubMed and Scopus databases for peer-reviewed research, review articles and clinical trials regarding GLP-1R agonists and PD published in the English language with no time restrictions. We also screened the references of the selected articles for possible additional articles in order to include most of the key recent evidence. Many data on animal models and preclinical studies show that GLP1-R agonists can restore dopamine levels, inhibit dopaminergic loss, attenuate neuronal degeneration and alleviate motor and non-motor features of PD. Evidence from clinical studies is also very promising, enhancing the possibility of adding GLP1-R agonists to the current armamentarium of drugs available for PD treatment.

Importance of beta cell mass for glycaemic control in people with type 1 diabetes.

AIMS/HYPOTHESIS: The role of beta cell mass in the balance of glucose control and hypoglycaemic burden in people with type 1 diabetes is unclear. We applied positron emission tomography (PET) imaging with radiolabelled exendin to compare beta cell mass among people with type 1 diabetes and either low glucose variability (LGV) or high glucose variability (HGV). METHODS: All participants with either LGV (n=9) or HGV (n=7) underwent a mixed-meal tolerance test to determine beta cell function and wore a blinded continuous glucose monitor for a week. After an i.v. injection with [68Ga]Ga-NODAGA-exendin-4, PET images were acquired for the quantification of pancreatic uptake of radiolabelled exendin. The mean standardised uptake value (SUVmean) of the pancreas was used to determine the amount of beta cell mass. RESULTS: Participants with LGV had lower HbA1c (46.0 mmol/mol [44.5-52.5] [6.4% (6.3-7)] vs 80 mmol/mol [69.0-110] [9.5% (8.5-12.2)], p=0.001) and higher time in range (TIR) (75.6% [73.5-90.3] vs 38.7% [25.1-48.5], p=0.002) than those with HGV. The SUVmean of the pancreas was higher for the LGV than for the HGV group (5.1 [3.6-5.6] vs 2.9 [2.1-3.4], p=0.008). The AUCC-peptide:AUCglucose ratio was numerically, but not statistically, higher in the LGV compared with the HGV group (2.7×10-2 [6.2×10-4-5.3×10-2] vs 9.3×10-4 [4.7×10-4-5.2×10-3], p=0.21). SUVmean correlated with the AUCC-peptide:AUCglucose ratio (Pearson r=0.64, p=0.01), as well as with the TIR (r=0.64, p=0.01) and the SD of interstitial glucose levels (r=-0.66, p=0.007). CONCLUSION/INTERPRETATION: Our data show higher beta cell mass in people with type 1 diabetes and LGV than in those with HGV, independent of beta cell function.

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.

New Radiolabeled Exendin Analogues Show Reduced Renal Retention.

PET imaging of the glucagon-like peptide-1 receptor (GLP-1R) using radiolabeled exendin is a promising imaging method to detect insulinomas. However, high renal accumulation of radiolabeled exendin could hamper the detection of small insulinomas in proximity to the kidneys and limit its use as a radiotherapeutic agent. Here, we report two new exendin analogues for GLP-1R imaging and therapy, designed to reduce renal retention by incorporating a cleavable methionine-isoleucine (Met-Ile) linker. We examined the renal retention and insulinoma targeting properties of these new exendin analogues in a nude mouse model bearing subcutaneous GLP-1R-expressing insulinomas. NOTA or DOTA was conjugated via a methionine-isoleucine linker to the C-terminus of exendin-4 (NOTA-MI-exendin-4 or DOTA-MI-exendin-4). NOTA- and DOTA-exendin-4 without the linker were used as references. The affinity for GLP-1R was determined in a competitive binding assay using GLP-1R transfected cells. Biodistribution of [68Ga]Ga-NOTA-exendin-4, [68Ga]Ga-NOTA-MI-exendin-4, [177Lu]Lu-DOTA-exendin-4, and [177Lu]Lu-DOTA-MI-exendin-4 was determined in INS-1 tumor-bearing BALB/c nude mice, and PET/CT was acquired to visualize renal retention and tumor targeting. For all tracers, dosimetric calculations were performed to determine the kidney self-dose. The affinity for GLP-1R was in the low nanomolar range (<11 nM) for all peptides. In vivo biodistribution revealed a significantly lower kidney uptake of [68Ga]Ga-NOTA-MI-exendin-4 at 4 h post-injection (p.i.) (34.2 ± 4.2 %IA/g), compared with [68Ga]Ga-NOTA-exendin-4 (128 ± 10 %IA/g). Accumulation of [68Ga]Ga-NOTA-MI-exendin-4 in the tumor was 25.0 ± 8.0 %IA/g 4 h p.i., which was similar to that of [68Ga]Ga-NOTA-exendin-4 (24.9 ± 9.3 %IA/g). This resulted in an improved tumor-to-kidney ratio from 0.2 ± 0.0 to 0.8 ± 0.3. PET/CT confirmed the findings in the biodistribution studies. The kidney uptake of [177Lu]Lu-DOTA-MI-exendin-4 was 39.4 ± 6.3 %IA/g at 24 h p.i. and 13.0 ± 2.5 %IA/g at 72 h p.i., which were significantly lower than those for [177Lu]Lu-DOTA-exendin-4 (99.3 ± 9.2 %IA/g 24 h p.i. and 45.8 ± 3.9 %IA/g 72 h p.i.). The uptake in the tumor was 7.8 ± 1.5 and 11.3 ± 2.0 %IA/g 24 h p.i. for [177Lu]Lu-DOTA-MI-exendin-4 and [177Lu]Lu-DOTA-exendin-4, respectively, resulting in improved tumor-to-kidney ratios for [177Lu]Lu-DOTA-MI-exendin-4. The new exendin analogues with a Met-Ile linker showed 2-3-fold reduced renal retention and improved tumor-to-kidney ratios compared with their reference without the Met-Ile linker. Future studies should demonstrate whether [68Ga]Ga-NOTA-MI-exendin-4 results in improved detection of small insulinomas in close proximity to the kidneys with PET/CT. [177Lu]Lu-DOTA-MI-exendin-4 might open a window of opportunity for exendin-based radionuclide therapy.

Targeted MicroRNA Profiling Reveals That Exendin-4 Modulates the Expression of Several MicroRNAs to Reduce Steatosis in HepG2 Cells.

Excess hepatic lipid accumulation is the hallmark of non-alcoholic fatty liver disease (NAFLD), for which no medication is currently approved. However, glucagon-like peptide-1 receptor agonists (GLP-1RAs), already approved for treating type 2 diabetes, have lately emerged as possible treatments. Herein we aim to investigate how the GLP-1RA exendin-4 (Ex-4) affects the microRNA (miRNAs) expression profile using an in vitro model of steatosis. Total RNA, including miRNAs, was isolated from control, steatotic, and Ex-4-treated steatotic cells and used for probing a panel of 799 highly curated miRNAs using NanoString technology. Enrichment pathway analysis was used to find the signaling pathways and cellular functions associated with the differentially expressed miRNAs. Our data shows that Ex-4 reversed the expression of a set of miRNAs. Functional enrichment analysis highlighted many relevant signaling pathways and cellular functions enriched in the differentially expressed miRNAs, including hepatic fibrosis, insulin receptor, PPAR, Wnt/ß-Catenin, VEGF, and mTOR receptor signaling pathways, fibrosis of the liver, cirrhosis of the liver, proliferation of hepatic stellate cells, diabetes mellitus, glucose metabolism disorder and proliferation of liver cells. Our findings suggest that miRNAs may play essential roles in the processes driving steatosis reduction in response to GLP-1R agonists, which warrants further functional investigation.

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.

Multivalent γ-PGA-Exendin-4 Conjugates to Target Pancreatic ß-Cells.

Targeting of glucagon-like peptide 1 receptor (GLP-1R), expressed on the surface of pancreatic ß-cells, is of great interest for the development of advanced therapies for diabetes and diagnostics for insulinoma. We report the conjugation of exendin-4 (Ex-4), an approved drug to treat type 2 diabetes, to poly-γ-glutamic acid (γ-PGA) to obtain more stable and effective GLP-1R ligands. Exendin-4 modified at Lysine-27 with PEG4-maleimide was conjugated to γ-PGA functionalized with furan, in different molar ratios, exploiting a chemoselective Diels-Alder cycloaddition. The γ-PGA presenting the highest number of conjugated Ex-4 molecules (average 120 per polymeric chain) showed a double affinity towards GLP-1R with respect to exendin per se, paving the way to improved therapeutic and diagnostic applications.

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.

Development of an 111In-Labeled Glucagon-Like Peptide-1 Receptor-Targeting Exendin-4 Derivative that Exhibits Reduced Renal Uptake.

Insulinomas are neuroendocrine tumors that are mainly found in the pancreas. Surgical resection is currently the first-line treatment for insulinomas; thus, it is vital to preoperatively determine their locations. The marked expression of the glucagon-like peptide-1 receptor (GLP-1R) is seen in pancreatic ß-cells and almost all insulinomas. Radiolabeled derivatives of exendin-4, a GLP-1R agonist, have been used with nuclear medicine imaging techniques for the in vivo detection of the GLP-1R; however, their marked renal accumulation can hinder the imaging of pancreatic tail lesions. To develop a GLP-1R imaging probe that exhibits reduced renal accumulation, we designed and synthesized a straight-chain GLP-1R-targeting radioligand, [111In]In-E4DA1, which consisted of exendin-4, DOTADG (a chelator), and an (iodophenyl)butyric acid derivative (an albumin binder [ALB]). We performed preclinical evaluations of [111In]In-E4DA1 to investigate its utility as a GLP-1R imaging probe. [111In]In-E4DA1 and [111In]In-E4D (a control compound lacking the ALB moiety) were prepared by reacting the corresponding precursors with [111In]InCl3 in buffer. Cell-binding and human serum albumin (HSA)-binding assays were performed to assess the in vitro affinity of the molecules for INS-1 (GLP-1R-positive) cells and albumin, respectively. A biodistribution assay and single-photon emission computed tomography imaging were carried out using INS-1 tumor-bearing mice. In the cell-binding assay, [111In]In-E4DA1 and [111In]In-E4D exhibited in vitro binding to INS-1 cells. In the HSA-binding assay, [111In]In-E4DA1 bound to HSA, while [111In]In-E4D showed little HSA binding. The in vivo experiments involving INS-1 tumor-bearing mice revealed that the introduction of an ALB moiety into the DOTADG-based exendin-4 derivative markedly increased the molecule's tumor accumulation while decreasing its renal accumulation. In addition, [111In]In-E4DA1 exhibited greater tumor accumulation than renal accumulation, whereas previously reported radiolabeled exendin-4 derivatives demonstrated much higher accumulation in the kidneys than in tumors. These results indicate that [111In]In-E4DA1 may be a useful GLP-1R imaging probe, as it demonstrates only slight renal accumulation.

Secretome-based screening of fusion partners and their application in recombinant protein secretion in Saccharomyces cerevisiae.

For the efficient production of heterologous proteins in the yeast Saccharomyces cerevisiae, we screened for a novel fusion partner from the yeast secretome. From twenty major proteins identified from the yeast secretome, we selected Scw4p, a cell wall protein with similarity to glucanase, and modified to develop a general fusion partner for the secretory expression of heterologous proteins in yeast. The optimal size of the SCW4 gene to act as an efficient fusion partner was determined by C-terminal truncation analysis; two of the variants, S1 (truncated at codon 115Q) and S2 (truncated at codon 142E), were further used for the secretion of heterologous proteins. When fused with S2, the secretion of three target proteins (hGH, exendin-4, and hPTH) significantly increased. Conserved O-glycosylation sites (Ser/Thr-rich domain) and hydrophilic sequences of S2 were deemed important for the function of S2 as a secretion fusion partner. Approximately 5 g/L of the S2-exendin-4 fusion protein was obtained from fed-batch fermentation. Intact target proteins were easily purified by affinity chromatography after in vitro processing of the fusion partner. This system may be of general application for the secretory production of heterologous proteins in S. cerevisiae. KEY POINTS : • Target proteins were efficiently secreted with their N-terminus fused to Scw4p. • O-glycosylation and hydrophilic stretches in Scw4p were important for protein secretion. • A variant of Scw4p (S2) was successfully applied for the secretory expression of heterologous proteins.

The glucagon-like peptide 1 receptor agonist Exendin-4 induces tenogenesis in human mesenchymal stem cells.

Tendon injuries are common and account for up to 50% of musculoskeletal injuries in the United States. The poor healing nature of the tendon is attributed to poor vascularization and cellular composition. In the absence of FDA-approved growth factors for tendon repair, engineering strategies using bioactive factors, donor cells, and delivery matrices to promote tendon repair and regeneration are being explored. Growth factor alternatives in the form of small molecules, donor cells, and progenitors offer several advantages and enhance the tendon healing response. Small drug molecules and peptides offer stability over growth factors that are known to suffer from relatively short biological half-lives. The primary focus of this study was to assess the ability of the exendin-4 (Ex-4) peptide, a glucagon-like peptide 1 (GLP-1) receptor agonist, to induce tenocyte differentiation in bone marrow-derived human mesenchymal stem cells (hMSCs). We treated hMSCs with varied doses of Ex-4 in culture media to evaluate proliferation and tendonogenic differentiation. A 20 nM Ex-4 concentration was optimal for promoting cell proliferation and tendonogenic differentiation. Tendonogenic differentiation of hMSCs was evaluated via gene expression profile, immunofluorescence, and biochemical analyses. Collectively, the levels of tendon-related transcription factors (Mkx and Scx) and extracellular matrix (Col-I, Dcn, Bgn, and Tnc) genes and proteins were elevated compared to media without Ex-4 and other controls including insulin and IGF-1 treatments. The tendonogenic factor Ex-4 in conjunction with hMSCs appear to enhance tendon regeneration.

The role of mast cells in cellular modifications evoked by Exendin-4 in treated wounds: a preclinical study.

OBJECTIVE: To assess the response of cellular infiltration in wounds treated with Exendin-4. METHOD: In this study, 16 mice were used. On each mouse, two wounds were produced, one above the other, in order to study the effects of the various treatments carried out. The wounds then received an intradermal injection of either saline (20µl; Group 1) or Exendin-4 (Exe4, 62ng; Group 2) in the upper and lower wounds, respectively. The mice were euthanised in order to collect the wounds at time of abrasion (T0), at 48 hours (T1), 96 hours (T2) and 144 hours (T3). The expression of the glucagon-like peptide-1 receptor (GLP-1R) was evaluated by Western blot in wound lysates. Histological and histochemistry methods were applied in cryosections. RESULTS: In T2 and T3 treated wounds, the mast cells degranulation index increased while GLP-1R expression, tumour necrosis factor (TNF)-α, or heat shock protein (HSP)47 antigens were detected in their cytoplasm. These cells interacted with dendritic cells, vessels or granulocytes. The density of dendritic cells increased progressively, and intercellular connections were found between these cells and vessels. Among the dendritic cells at T2, only M2 macrophages increased. However, M1 cells expressed transforming growth factor (TGF)-ß and both interacted with either fibroblasts or with vessels. The number of plasmacytoid dendritic cells increased and established close contacts with regulatory T cells. CONCLUSION: We propose that after treatment with Exe4, early activation of mast cells is critical in wound healing acceleration. This is crucial in understanding the potential effect of this drug for viable clinical therapies. DECLARATION OF INTEREST: No potential conflict of interest was reported by the authors.

Targeting Persistent Neuroinflammation after Hypoxic-Ischemic Encephalopathy-Is Exendin-4 the Answer?

Hypoxic-ischemic encephalopathy is brain injury resulting from the loss of oxygen and blood supply around the time of birth. It is associated with a high risk of death or disability. The only approved treatment is therapeutic hypothermia. Therapeutic hypothermia has consistently been shown to significantly reduce the risk of death and disability in infants with hypoxic-ischemic encephalopathy. However, approximately 29% of infants treated with therapeutic hypothermia still develop disability. Recent preclinical and clinical studies have shown that there is still persistent neuroinflammation even after treating with therapeutic hypothermia, which may contribute to the deficits seen in infants despite treatment. This suggests that potentially targeting this persistent neuroinflammation would have an additive benefit in addition to therapeutic hypothermia. A potential additive treatment is Exendin-4, which is a glucagon-like peptide 1 receptor agonist. Preclinical data from various in vitro and in vivo disease models have shown that Exendin-4 has anti-inflammatory, mitochondrial protective, anti-apoptotic, anti-oxidative and neurotrophic effects. Although preclinical studies of the effect of Exendin-4 in perinatal hypoxic-ischemic brain injury are limited, a seminal study in neonatal mice showed that Exendin-4 had promising neuroprotective effects. Further studies on Exendin-4 neuroprotection for perinatal hypoxic-ischemic brain injury, including in large animal translational models are warranted to better understand its safety, window of opportunity and effectiveness as an adjunct with therapeutic hypothermia.

Development and Validation of an Analytical HPLC Method to Assess Chemical and Radiochemical Purity of [68Ga]Ga-NODAGA-Exendin-4 Produced by a Fully Automated Method.

Background: Glucagon-like peptide 1 receptor (GLP-1R) is preferentially expressed in pancreatic islets, especially in ß-cells, and highly expressed in human insulinomas and gastrinomas. In recent years several GLP-1R-avid radioligands have been developed to image insulin-secreting tumors or to provide a tentative quantitative in vivo biomarker of pancreatic ß-cell mass. Exendin-4, a 39-amino acid peptide with high binding affinity to GLP-1R, has been labeled with Ga-68 for imaging with positron emission tomography (PET). Preparation conditions may influence the quality and in vivo behavior of tracers. Starting from a published synthesis and quality controls (QCs) procedure, we have developed and validated a new rapid and simple UV-Radio-HPLC method to test the chemical and radiochemical purity of [68Ga]Ga-NODAGA-exendin-4, to be used in the clinical routine. Methods: Ga-68 was obtained from a 68Ge/68Ga Generator (GalliaPharma®) and purified using a cationic-exchange cartridge on an automated synthesis module (Scintomics GRP®). NODAGA-exendin-4 contained in the reactor (10 µg) was reconstituted with HEPES and ascorbic acid. The reaction mixture was incubated at 100 °C. The product was purified through HLB cartridge, diluted, and sterilized. To validate the proposed UV-Radio-HPLC method, a stepwise approach was used, as defined in the guidance document released by the International Conference on Harmonization of Technical Requirements of Pharmaceuticals for Human Use (ICH), adopted by the European Medicines Agency (CMP/ICH/381/95 2014). The assessed parameters are specificity, linearity, precision (repeatability), accuracy, and limit of quantification. Therefore, a range of concentrations of Ga-NODAGA-exendin-4, NODAGA-exendin-4 (5, 4, 3.125, 1.25, 1, and 0.75 µg/mL) and [68Ga]Ga-NODAGA-exendin-4 were analyzed. To validate the entire production process, three consecutive batches of [68Ga]Ga-NODAGA-exendin-4 were tested. Results: Excellent linearity was found between 5-0.75 µg/mL for both the analytes (NODAGA-exendin-4 and 68Ga-NODAGA-exendin-4), with a correlation coefficient (R2) for calibration curves equal to 0.999, average coefficients of variation (CV%) < 2% (0.45% and 0.39%) and average per cent deviation value of bias from 100%, of 0.06% and 0.04%, respectively. The calibration curve for the determination of [68Ga]Ga-NODAGA-exendin-4 was linear with a R2 of 0.993 and CV% < 2% (1.97%), in accordance to acceptance criteria. The intra-day and inter-day precision of our method was statistically confirmed using 10 µg of peptide. The mean radiochemical yield was 45 ± 2.4% in all the three validation batches of [68Ga]Ga-NODAGA-exendin-4. The radiochemical purity of [68Ga]Ga-NODAGA-exendin-4 was >95% (97.05%, 95.75% and 96.15%) in all the three batches. Conclusions: The developed UV-Radio-HPLC method to assess the radiochemical and chemical purity of [68Ga]Ga-NODAGA-exendin-4 is rapid, accurate and reproducible like its fully automated production. It allows the routine use of this PET tracer as a diagnostic tool for PET imaging of GLP-1R expression in vivo, ensuring patient safety.

The metabolite GLP-1 (9-36) is neuroprotective and anti-inflammatory in cellular models of neurodegeneration.

Glucagon-like peptide-1 (GLP-1) is best known for its insulinotropic action following food intake. Its metabolite, GLP-1 (9-36), was assumed biologically inactive because of low GLP-1 receptor (GLP-1R) affinity and non-insulinotropic properties; however, recent studies contradict this assumption. Increased use of FDA approved GLP-1 analogues for treating metabolic disorders and neurodegenerative diseases raises interest in GLP-1 (9-36)'s biological role. We use human SH-SY5Y neuroblastoma cells and a GLP-1R over-expressing variety (#9), in both undifferentiated and differentiated states, to evaluate the neurotrophic/neuroprotective effects of GLP-1 (9-36) against toxic glutamate exposure and other oxidative stress models (via the MTS, LDH or ROS assays). In addition, we examine GLP-1 (9-36)'s signaling pathways, including cyclic-adenosine monophosphate (cAMP), protein kinase-A (PKA), and 5' adenosine monophosphate-activated protein kinase (AMPK) via the use of ELISA, pharmacological inhibitors, or GLP-1R antagonist. Human HMC3 and mouse IMG microglial cell lines were used to study the anti-inflammatory effects of GLP-1 (9-36) against lipopolysaccharide (LPS) (via ELISA). Finally, we applied GLP-1 (9-36) to primary dissociation cultures challenged with α-synuclein or amyloid-ß and assessed survival and morphology via immunochemistry. We demonstrate evidence of GLP-1R, cAMP, PKA, and AMPK-mediated neurotrophic and neuroprotective effects of GLP-1 (9-36). The metabolite significantly reduced IL-6 and TNF-α levels in HMC3 and IMG microglial cells, respectively. Lastly, we show mild but significant effects of GLP-1 (9-36) in primary neuron cultures challenged with α-synuclein or amyloid-ß. These studies enhance understanding of GLP-1 (9-36)'s effects on the nervous system and its potential as a primary or complementary treatment in pathological contexts.