Stimulated Insulin Release Research Articles

Profiling and activity screening of Dammarane-type triterpen saponins from Gynostemma pentaphyllum with glucose-dependent insulin secretory activity

The global prevalence of type 2 diabetes is increasing rapidly; consequently there is great need for new and novel therapeutic options. Gynostemma pentaphyllum (GP) is a traditional medicinal plant, mainly present in Southeast Asian countries, that has been reported to exert antidiabetic effects, by stimulating insulin secretion. The specific compound responsible for this effect is however as yet unidentified. Screening for discovery and identification of bioactive compounds of an herbal GP extract, was performed in isolated pancreatic islets from spontaneously diabetic Goto-Kakizaki (GK) rats, a model of type 2 diabetes, and from non-diabetic control Wistar rats. From this herbal extract 27 dammarane-type saponins, including two novel compounds, were isolated and their structure was elucidated by mass spectrometry and NMR spectroscopy. One of the dammarane-type triterpenoid showed a glucose-dependent insulin secretion activity. This compound, gylongiposide I, displays unique abilities to stimulate insulin release at high glucose levels (16.7 mM), but limited effects at a low glucose concentration (3.3 mM). Further studies on this compound, also in vivo, are warranted with the aim of developing a novel anti-diabetic therapeutic with glucose-dependent insulinogenic effect.

Peptidomic analysis of the host-defense peptides in skin secretions of Rana graeca provides insight into phylogenetic relationships among Eurasian Rana species

Peptidomic analysis of norepinephrine-stimulated skin secretions from the Greek stream frog Rana graeca Boulenger, 1891 led to the identification and structural characterization of a range of host-defense peptides. These comprised brevinin-1GRa, brevinin-1GRb and an N-terminally extended form of brevinin-1GRb, ranatuerin-2GR together with its oxidized form and (11–28) fragment, temporin-GRa, temporin-GRb and its non-amidated form, and a melittin-related peptide, MRP-GR and its (1–18) fragment. The most abundant peptide, MRP-GR significantly (P < 0.001) stimulated insulin release from BRIN-BD11 clonal β-cells at concentrations ≥0.1 nM. Rana graeca (formerly Rana graeca graeca) and the morphologically similar Italian stream frog Rana italica Dubois, 1987 (formerly Rana graeca italica) were originally regarded as sub-species. However, the primary structures of the host defense peptides from both frogs support the claim based upon comparisons of the nucleotide sequences of S1 satellite DNA that R. graeca and R. italica are separate species. Cladistic analyses based upon the primary structures of the brevinin-1 and ranatuerin-2 peptides from Eurasian frogs indicate a close phylogenetic relationship between R. graeca and Rana latastei whereas R. italica is most closely related to Rana dalmatina.

Abscisic acid stimulates the release of insulin and of GLP‐1 in the rat perfused pancreas and intestine

Previous results indicate that nanomolar concentrations of abscisic acid (ABA) stimulate insulin release from β-pancreatic cells in vitro and that oral ABA at 50mg/kg increases plasma GLP-1 in the fasted rat. The aim of this study was to test the effect of ABA on the perfused rat pancreas and intestine, to verify the insulin- and incretin-releasing actions of ABA in controlled physiological models. Rat pancreas and small intestine were perfused with solutions containing ABA at high-micromolar concentrations, or control secretagogues. Insulin and GLP-1 concentrations in the venous effluent were analysed by radioimmunoassay, and ABA levels were determined by ELISA. High micromolar concentrations of ABA induced GLP-1 secretion from the proximal half of the small intestine and insulin secretion from pancreas. GLP-1 stimulated ABA secretion from pancreas in a biphasic manner. Notably, a positive correlation was found between the ABA area under the curve (AUC) and the insulin AUC upon GLP-1 administration. Our results indicate the existence of a cross talk between GLP-1 and ABA, whereby ABA stimulates GLP-1 secretion, and vice versa. Release of ABA could be considered as a new promising molecule in the strategy of type 2 diabetes treatment and as a new endogenous hormone in the regulation of glycaemia.

Multilineage potential research on pancreatic mesenchymal stem cells of bovine.

Stem cells are most likely to solve all three of diabetes's problems at once, but the previous studies have mostly focused on bone marrow mesenchymal stem cells (MSCs) and adipose tissue-derived MSCs, and few studies have been done on pancreatic MSCs. In this study, pancreatic was collected to isolate MSCs from bovine, and then their biological characteristics such as growth kinetics, surface antigen, and multilineage potential were examined. Pancreatic MSCs of bovine (B-PMSCs) could be cultured for 65 passages in vitro. Growth kinetics analyses indicated that B-PMSCs had a strong capacity for self-renewal in vitro and their proliferation capacity appeared to decrease by passaging. Surface antigen detection showed that B-PMSCs expressed CD29, CD44, CD73, CD90, CD106, CD166, Vimentin, Nestin and Insulin, but not expressed CD34 and CD45. Furthermore, B-PMSCs could be induced to differentiate into adipocytes, osteoblasts and smooth muscle cells as indicated by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence. Most importantly, insulin-secreting cell differentiation of B-PMSCs exhibited islet-like clusters and dithizone staining displayed scarlet, and the response of the islet-like clusters to glucose suggested that high concentration glucose (20 mM) could quickly and persistently stimulate insulin release, and from the 2.0 h of the stimulation, the insulin of 20 mM glucose group were significantly higher than the 5.5 mM group. The B-PMSCs were isolated successfully, and the cells owned powerful self-renewal ability and multiple differentiative potential. Therefore, the present study plays an important role by providing a PMSCs choice for cell therapy of diabetes and tissue engineering.

Advances in Human Islet Processing: Manufacturing Steps to Achieve Predictable Islet Outcomes from Research Pancreases

Background This presentation of a six-year study processing human islets for research and transplantation includes a review of multi-center transplant studies identifying key variables critical for successful islet processing and defines standardized processing procedures required to provide highly purified, functional Human Islets. Methods Human islet processing methods are defined in detail with pancreas retrieval, shipping, trimming for processing, collagenase distension, controlled digestion by digestion/filtration method, islet purification and islet culture. Islet processing results are summarized from 27 published reports (2003-2017) from 21 international clinical islet transplant centers with 13 single islet centers and 8 from multi-center clinical trials that averaged islet yields of 5,680 IEQ/Gm (Pre-Purification), 4,101 IEQ/Gm (Post-Purification), and 3,599 IEQ/Gm (Post-Culture) with 59.2% purity at time of islet transplant into the liver. Results Their results were compared to this study of 226 Non-DM (Non-Diabetes Mellitus) donor processing islet yields that averaged 6,942.7±201 IEQ/Gm (Pre-Purification), 5,484.5±199 IEQ/Gm (Post-Purification), and 4,351.0±167 IEQ/Gm (Post-Culture) per pancreas with 90.0% purity at time of islet distribution. Islet processing from 29 Type 2 Diabetes donors resulted in reduced islet yields of 5,797±734 IEQ/Gm (Pre-Purification), 4,371±866 IEQ/Gm (Post-Purification), and 3,323±423 IEQ/Gm (Post-Culture) with similar purity but reduced insulin content. Glucose Stimulated Insulin Release testing in T2D islets showed significantly reduced insulin release at 20mM and 20mM+IBMX versus Non-DM islets and showed significantly reduced Total Stimulated Insulin Release of 0.722±0.142 (T2D) versus 1.069±0.067 ng insulin/ng DNA (Non-DM). Conclusions Of the significant process variables, short Switch Times were predominant in greatly increasing islet yields, GSIR results, and insulin content with any time

Glucagon-related peptides from phylogenetically ancient fish reveal new approaches to the development of dual GCGR and GLP1R agonists for type 2 diabetes therapy

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.

Improved recovery of human islets from young donor pancreases utilizing increased protease dose to collagenase for digesting peri-islet extracellular matrix.

Human islet isolation from young donor pancreases (YDP) utilizing the current purified standard dose of collagenase-protease enzyme mixtures often results in the release of a high percentage of mantled islets. Mantled islets are those surrounded by exocrine tissue and are difficult to purify by density gradient centrifugation, leading to poor islet recovery. Based on difference in extracellular matrix, and total collagen content between YDP and old donor pancreas (ODP, > 35 Y) led us to compare results from islet isolation using increased collagenase combination (ICC) or increased protease combination (IPC), to the standard enzyme combination (SEC) in a "trisected" pancreas model to overcome the donor-to-donor variability. These results showed a reduced percentage of mantled islets (17%±7.5%) and higher postpurification islet recovery (83.8%±5.6%) with IPC. Furthermore, these results were confirmed in 13 consecutive whole pancreas islet isolations utilizing IPC from VitaCyte, Roche, or SERVA collagenase-protease enzyme mixtures. Results obtained from in vitro and in vivo islet functional assessment indicated that islets isolated using IPC retained normal islet morphology, insulin secretion, and the ability to reverse diabetes after transplantation in diabetic nude mice. This is the first report utilizing trisected pancreas to assess the effectiveness of different enzyme combinations to improve islet recovery from young donor pancreases.

Effects of dexmedetomidine and MK-467 on plasma glucose, insulin and glucagon in a glibenclamide-induced canine hypoglycaemia model

The commonly used sedative α2-adrenoceptor agonist dexmedetomidine has adverse cardiovascular effects in dogs that can be prevented by concomitant administration of the peripherally acting α2-adrenoceptor antagonist MK-467. An ancillary effect of dexmedetomidine is to decrease insulin release from the pancreas, whereas MK-467 stimulates insulin release. This study assessed the effects of co-administered dexmedetomidine and MK-467 in a canine glibenclamide-induced hypoglycaemia model. In a randomised, cross-over experiment, eight beagle dogs received five intravenous treatments, comprising two administrations of saline, with dexmedetomidine or dexmedetomidine and MK-467, and three administrations of glibenclamide, with saline, dexmedetomidine or dexmedetomidine and MK-467. Plasma concentrations of glucose, lactate, insulin, glucagon and the test drugs were monitored. Administration of glibenclamide significantly increased insulin secretion and decreased blood glucose concentrations. Dexmedetomidine counteracted glibenclamide-evoked hypoglycaemia. This was opposed by the α2-adrenoceptor antagonist MK-467, but the glibenclamide-evoked hypoglycaemia was not potentiated by co-administration of dexmedetomidine and MK-467. None of the dogs developed uncontrolled hypoglycaemia. Thus, the combination of dexmedetomidine and MK-467 appeared to be safe in this canine hypoglycaemia model. Nevertheless, when MK-467 is used to alleviate the undesired cardiovascular effects of α2-adrenoceptor agonists in dogs, it should be used with caution in animals at risk for hypoglycaemia because of its insulin-releasing and hypoglycaemic effects.

Insulinotropic activity of the host-defense peptide frenatin 2D: Conformational, structure-function and mechanistic studies

Of four naturally occurring frenatin peptides tested, frenatin 2D (DLLGTLGNLPLPFI.NH2) from Discoglossus sardus was the most potent and effective in producing concentration-dependent stimulation of insulin release from BRIN-BD11 rat clonal β-cells without displaying cytotoxicity. The peptide also stimulated insulin release from 1.1B4 human-derived clonal β-cells and isolated mouse islets and improved glucose tolerance concomitant with increased circulating insulin concentrations in mice following intraperitoneal administration. The insulinotropic activity of frenatin 2D was not associated with membrane depolarization or an increase in intracellular [Ca2+] but incubation of the peptide (1 μM) with BRIN-BD11 cells produced a modest, but significant (P < 0.05), increase in cAMP production. Stimulation of insulin release was abolished in protein kinase A-downregulated cells but maintained in protein kinase C-downregulated cells. Circular dichroism studies showed that, in the presence of dodecylphosphocholine micelles, frenatin 2D exhibited a helical content of 35% and a turn content of 28%. Substitution of the Thr5, Asn8, Pro10, and Ile14 residues in frenatin-2D by Trp and interchange of Pro12 and Phe13 led to loss of insulinotropic activity but the [D1W] and [G7W] analogues were as potent and effective as the native peptide. Frenatin 2D (1 μM) also stimulated proliferation of BRIN-BD11 cells and provided significant protection of the cells against cytokine-induced apoptosis. It is concluded that the insulinotropic activity of frenatin 2D is mediated predominantly, if not exclusively, by the KATP channel-independent pathway.

Fatty Acids and Insulin Secretion: From FFAR and Near?

Nonesterified, long-chain fatty acids (LCFAs) potentiate glucose-stimulated insulin secretion (GSIS). In other words, LCFAs do not trigger insulin release by themselves but strongly amplify the stimulating effect of glucose (1). Yet, from a physiological standpoint it does not seem logical that an energetic substrate whose circulating levels are elevated when glucose levels are low would stimulate insulin release (2). This apparent contradiction can be resolved if the LCFAs that stimulate insulin secretion come from the local islet environment, where their concentrations are likely significantly higher than plasma levels, rather than from the circulation (3). LCFA potentiation of GSIS occurs via at least two distinct mechanisms. LCFAs are transported across the plasma membrane, activated into long-chain acyl-CoA, and metabolized to generate lipid signaling molecules that promote insulin exocytosis when glucose levels are elevated (3). LCFAs also bind to and activate the G-protein–coupled receptor FFAR1 (GPR40), which is mainly coupled to Gαq/11 and triggers a signaling cascade leading to the activation of protein kinase D1 and insulin release (4) (Fig. 1). Intracellular metabolism and FFAR1 activation each account for approximately 50% of the overall effect of exogenous LCFA on insulin release (5). The question of how much LCFA is available at the vicinity of the islet for uptake or FFAR1 activation is complex. First, the vast majority of LCFA in the circulation and interstitial space is bound to albumin, and the bioactive, non-BSA–bound fraction is extremely small (6). Second, lipoprotein lipase expressed in islet capillaries delivers LCFA from postprandial chylomicrons to the β-cell (7). Third, β-cells exhibit significant glucose-stimulated lipolytic activity (8,9), which …

On the effects of NLRP3 inflammasome on mice pancreatic β-cell damage induced by advanced glycation end products

Objective To explore the role of the pyrin domain-containing 3 (NLRP3) inflammasome in advanced glycation end products (AGEs)-induced mice pancreatic β-cell damage. Methods AGEs were administered intraperitoneally for 6 weeks in NLRP3 knockout mice or C57BL/6J mice. Intraperitoneal glucose tolerance test and insulin releasing test were performed. Pancreatic sections were stained with haematoxylin and eosin, or with F4/80 and NLRP3 antibodies. Insulin and pancreatic tissue monocyte chemotactic protein 1 (MCP-1) as well as interleukin-1β (IL-1β) levels were measured with ELISA kits. Expression of MCP-1 protein was determined by western blot. MIN6 cells and mouse peritoneal macrophages cells were treated with AGEs and different interventions (antioxidant NAC, adenovirus NLRP3 shRNA or NLRP3 knockout). Reactive oxygen species production, NLRP3 mRNA expression, IL-1β secretion, caspase 1 activity, apoptosis and glucose stimulated insulin release were determined. Results Injection of AGEs induced an abnormal response to glucose, enhanced the insulitis score, and increased the levels of pancreatic tissue MCP-1 and IL-1β, as well as raised the expression of NLRP3 and F4/80 in pancreatic islet. Remarkably, co-localization of NLRP3 and macrophage marker F4/80 was observed in islet. The damages were improved in NLRP3 knockout mice. After incubation with AGEs, reactive oxygen species production and cell apoptosis was enhanced, NLRP3 inflammasome activated, with glucose-stimulated insulin release impaired in MIN6 cells. NAC treatment alliviated the above damages, but NLRP3 gene silencing had no effect on ROS level, apoptosis, and insulin secretion. Finally NAC treatment and NLRP3 gene knockout inhibited activation of NLRP3 inflammasome induced by AGEs in mouse peritoneal macrophages cells. Conclusion NLRP3 knockout ameliorates the islet β-cell damage induced by AGEs. These effects were associated with AGEs-induced islets macrophage infiltrating by up-regulation of MCP-1 expression, and AGEs-induced activation of NLRP3 inflammasome in macrophage through ROS pathway, which results in the release of active IL-1β and leads to the lesions of β-cell. (Chin J Endocrinol Metab, 2018, 34: 690-695) Key words: Advanced glycation end products; NLRP3 inflammasome; β-cell damage

Effect of nociceptin on insulin release in normal and diabetic rat pancreas.

Nociceptin (NC), also known as Orphanin FQ, is a brain peptide involved in the regulation of pain, but its role in the endocrine pancreas is poorly understood. The present study examines the pattern of distribution of NC and its effect on insulin and glucagon secretion after the onset of diabetes mellitus (DM). Male Wistar rats weighing 150-200g were made diabetic with streptozotocin (60mg/kg body weight, intraperitoneally). Four weeks after the induction of DM, pancreatic tissues were retrieved and processed for immunofluorescence, immunoelectron microscopy, and insulin and glucagon secretion. Isolated islets from non-diabetic and diabetic rats were used to determine the effect of NC on insulin release. NC was discerned in islet cells of non-diabetic control and diabetic rat pancreata. NC co-localized only with insulin in pancreatic beta cells. NC did not co-localize with either glucagon or somatostatin or pancreatic polypeptide. The number of NC-positive cells was markedly (p < 0.001) reduced after the onset of DM. Electron microscopy study showed that NC is located with insulin in the same secretory granules of the beta cells of both non-diabetic and diabetic rat pancreas. NC inhibits insulin release markedly (p < 0.05) from pancreatic tissue fragments of non-diabetic and diabetic rats. In contrast, NC at 10-12M stimulates insulin release in isolated islets of DM rats. In conclusion, NC co-localizes with insulin only in the islet of Langerhans. The co-localization of NC with insulin suggests a role for NC in the regulation of pancreatic beta cell function.

DPP-IV Inhibitory Potentials of Flavonol Glycosides Isolated from the Seeds of Lens culinaris: In Vitro and Molecular Docking Analyses.

Dipeptidyl peptidase IV (DPP-IV), a new target for the treatment of type 2 diabetes mellitus, degrades incretins such as glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide. DPP-IV inhibitors shorten the inactivation of GLP-1, permitting the incretin to stimulate insulin release, thereby combating hyperglycemia. In our ongoing search for new DPP-IV inhibitors from medicinal plants and foods, three flavonol glycosides (1–3) were isolated from the seeds of Lens culinaris Medikus (Fabaceae) and tested for their DPP-IV–inhibitory activity. We demonstrated for the first time, that compounds 1–3 inhibited DPP-IV activity in a concentration-dependent manner in our in vitro bioassay system. In addition, molecular docking experiments of compounds 1–3 within the binding pocket of DPP-IV were conducted. All investigated compounds readily fit within the active sites of DPP-IV, in low-energy conformations characterized by the flavone core structure having optimal electrostatic attractive interactions with the catalytic triad residues of DPP-IV.

Lupinus mutabilis Extract Exerts an Anti-Diabetic Effect by Improving Insulin Release in Type 2 Diabetic Goto-Kakizaki Rats.

Lupinus mutabilis (LM) is a legume part of Bolivian traditional diet that has a nutraceutical property reducing blood glucose levels. The prevalence of type 2 diabetes is increasing worldwide thus; the search for novel anti-diabetic drugs is needed. Based on its traditional use, we evaluated the anti-diabetic effect of LM in the spontaneously diabetic Goto-Kakizaki (GK) rat, a model of type 2 diabetes and in Wistar (W) rats as healthy control. LM seeds hydroethanolic extract, analyzed by gas chromatography-mass spectrometry and high-performance liquid chromatography-high resolution mass spectrometry, is a complex mixture of volatile and non-volatile components. A single oral administration of LM extract (2000 mg/kg b.w.) improved glucose tolerance during the oral glucose tolerance test (OGTT) (30–120 min) in GK and W rats (p < 0.0001). The long-term treatment with LM (1000 mg/kg b.w.), for 21 days, improved the area under the curve (AUC) of glucose during OGTT at day 20, in both GK (p < 0.01) and W rats (p < 0.01). The HbA1c (GK rats, p < 0.05 and W rats, p < 0.0001) and the non-fasting glucose (GK rats, p < 0.05) were also reduced. LM increased both serum insulin levels (2.4-fold in GK rats and 2.5-fold W rats), and the glucose-induced (16.7 mM glucose) insulin release in isolated islets from treated animals (6.7-fold in GK rats, and 6.6-fold in W rats). Moreover, LM (10 mg/mL) stimulated in vitro glucose induced (16.7 mM glucose) insulin release in batch incubated GK and W rat islets (p < 0.0001). In perifused GK rat islets, insulin release in 16.7 mM glucose was increased 95.3-fold compared to untreated islets (p < 0.0001), while no significant differences were found in perifused W rat islets. The LM mechanism of action, evaluated using inhibitory compounds of the insulin secretion pathway, showed that LM-dependent insulin secretion was reduced 42% by diazoxide (p < 0.001), 70% by nifedipine (p < 0.001), 86.7% by H89 (p < 0.0001), 70.8% by calphostine-C (p < 0.0001) and 93% by pertussis toxin (p < 0.0001). A similar effect was observed in W rats islets. Our findings provide evidence that LM has an anti-diabetic effect through stimulation of insulin release. The effect is-dependent on L-type calcium channel, protein kinase A and C systems, and G protein-coupled exocytosis and is partially mediated by K-ATP channels.

Ultrasound-Induced Insulin Release as a Potential Novel Treatmentfor Type 2 Diabetes Mellitus.

Therapeutic ultrasound presents a potential novel treatment for type 2 diabetes mellitus that utilizes the non-invasive application of ultrasound energy to treat secretory defects in the earlier stages of the disease. Our previous studies have shown that ultrasound is capable of stimulating insulin release from pancreatic beta cells, safely and effectively. This study aims to both examine the calcium-dependent mechanisms of ultrasound-mediated insulin release from pancreatic beta cells using three complementary modalities - carbon fiber amperometry, ELISA studies, and Ca2+ fluorescence imaging - and to study the translational potential of therapeutic ultrasound using transgenic hyperglycemic mice for safety and efficacy studies.

Sodium-glucose Cotransporter 2 Inhibitors and Ischemic Stroke.

Type 2 diabetes mellitus (T2DM) is associated with substantially increased risk for cardiovascular events, including ischemic stroke. In turn, ischemic stroke represents a leading cause of mortality and long-term disability worldwide. The recent class of glucose-lowering agents is sodium-glucose cotransporter 2 (SGLT-2) inhibitors, which act through inhibition of glucose reabsorption in the kidney, resulting in glucose excretion without stimulating insulin release. Accumulating data suggests that these agents improve multiple risk factors for ischemic stroke except their glucose-lowering effect. In the present review, the pleiotropic actions of SGLT-2 inhibitors are summarized and their potential implications on ischemic stroke prevention are discussed. We performed a comprehensive search of the literature in terms of SGLT-2 inhibitors efficacy on ischemic stroke and traditional risk factors of cerebrovascular disease. Several studies consistently showed that SGLT-2 inhibitors reduce blood pressure, induce weight loss, increase high-density lipoprotein cholesterol levels and reduce triglyceride levels. In addition, they improve several emerging cardiovascular risk factors, most notably arterial stiffness, albuminuria and oxidative stress. However, in the only trial that evaluated the effects of these agents on the incidence of ischemic stroke, empagliflozin did not reduce the risk of first or recurrent stroke despite a significant reduction in cardiovascular and all-cause mortality. Despite the multiple pleiotropic effects of SGLT-2 inhibitors, these agents do not appear to affect stroke risk. Ongoing large trials with longer follow-up will evaluate whether the pleiotropic effects of this class will translate into benefits in ischemic stroke prevention.

Functional Maturation of Pancreatic ß Cells during Development

Insulin is crucial for intrauterine growth of the fetus and insulin secretion must be maintained irrespective of maternal plasma glucose variations. This is achieved through a maturational islet adaptation which persists during the first postnatal days and can lead to prolonged neonatal hypoglycemia. We aimed to investigate the characteristics of insulin release in the fetal and perinatal period. We performed perifusions of human fetal islet samples at 18-20 weeks gestation and of rat islets between embryonic day (e)22 and postnatal day (P)14. Human fetal islets at 18-20 weeks gestation released insulin in response to lower glucose concentrations (1-2 mM), compared to adult islets (3-5 mM). Similarly, rat islets at e22-P1 also had a lower glucose threshold for insulin release (3mM), compared to P14 islets (5 mM). BCH, a leucine analog that activates glutamate dehydrogenase, also stimulated insulin release at a lower concentration in e22-P1 rat islets (1mM), compared to P14 islets (3mM). This suggests that the mechanism of the islet adaptation in intrauterine life lies distal to glycolysis and fuel oxidation, potentially in differences in membrane depolarization or repolarization. In addition, human fetal islets released insulin in response to lactate, while late embryonic rat islets did not, suggesting insulin release in response to other nutrient stimuli may also be part of this maturational islet adaptation. Ongoing experiments are further exploring substrate-induced insulin release in immature islets, in parallel with the characterization of β cell transcriptomic maturation at the single cell level in both human and rat. Understanding the functional maturation of β cells during the fetal and neonatal period, coupled with the insight into the molecular mechanisms responsible for this maturation, can lead of the development of molecular markers of a dysregulated β cell function that is may persist beyond early neonatal period. Disclosure D.E. Stanescu: Employee; Spouse/Partner; GlaxoSmithKline plc.. C. Li: None. J. Yang: None. D. Yau: None. C.A. Stanley: None.

A Positive Allosteric Modulator Enhances the Ability of Beta Cell M3 Muscarinic Receptors to Simulate Insulin Secretion In Vitro and In Vivo

Several studies have shown that activation of M3 muscarinic acetylcholine (ACh) receptors (M3Rs) expressed by pancreatic beta-cells leads to striking increases in glucose-stimulated insulin release. As a result, we hypothesized that agents that can promote signaling through beta-cell M3Rs might become useful in the treatment of type 2 diabetes. The M3R is a prototypic class A GPCR that is selectively coupled to G proteins of the Gq family. At present, selective M3R agonists that bind to the orthosteric ACh binding site are not available. However, the lab of Craig Lindsley (Bridges et al., 2010) recently described a compound (VU0119498) that binds to an allosteric site on the M3R, thus enhancing ACh-induced signaling through M3Rs. In this study, we tested the ability of this positive allosteric modulator (PAM) to stimulate insulin release in vitro and in vivo. The use of PAMs has the great advantage that it respects the spatio-temporal control of receptor activation, i.e., the PAM is only active upon receptor occupation by an endogenous orthosteric agonist. Studies with cultured MIN6 cells showed that VU0119498 enhanced ACh-induced insulin secretion in an M3R-dependent fashion. In vivo studies demonstrated that PAM treatment of WT mice caused a significant increase in plasma insulin levels, accompanied by a striking improvement in glucose tolerance. These VU0119498 effects were mediated by beta-cell M3Rs since they were absent in mutant mice selectively lacking M3Rs in beta-cells. In future studies, we will use mouse models of diabetes to examine whether this M3R-PAM is endowed with antidiabetic activity. This approach may lead to the development of a novel generation of antidiabetic drugs. Disclosure L. Zhu: None. J. Wess: None.

Xylitol toxicosis in dogs

Xylitol is a commonly available sugar substitute found in sugar-free chewing gums and as an excipient in some medicines. It is used for its benefits on oral hygiene and its low-calorie content. It is also found in some peanut butters, ice creams and toothpastes, and can be used as a sugar substitute in baking. Xylitol causes hypoglycaemia (due to stimulation of insulin release) and liver failure in dogs, but not in cats, rabbits or rodents. The hypoglycaemia may be rapid in onset but can be delayed if xylitol-containing chewing gum has been ingested. Management of dogs that have ingested xylitol includes gut decontamination, monitoring, correction of hypoglycaemia and liver protectants. Prognosis is good in most cases and death from profound, uncontrolled hypoglycaemia or liver failure is not common.

Secretagogin is increased in plasma from type 2 diabetes patients and potentially reflects stress and islet dysfunction.

Beta cell dysfunction accompanies and drives the progression of type 2 diabetes mellitus (T2D), but there are few clinical biomarkers available to assess islet cell stress in humans. Secretagogin, a protein enriched in pancreatic islets, demonstrates protective effects on beta cell function in animals. However, its potential as a circulating biomarker released from human beta cells and islets has not been studied. In this study primary human islets, beta cells and plasma samples were used to explore secretion and expression of secretagogin in relation to the T2D pathology. Secretagogin was abundantly and specifically expressed and secreted from human islets. Furthermore, T2D patients had an elevated plasma level of secretagogin compared with matched healthy controls, which was confirmed in plasma of diabetic mice transplanted with human islets. Additionally, the plasma secretagogin level of the human cohort had an inverse correlation to clinical assessments of beta cell function. To explore the mechanism of secretagogin release in vitro, human beta cells (EndoC-βH1) were exposed to elevated glucose or cellular stress-inducing agents. Secretagogin was not released in parallel with glucose stimulated insulin release, but was markedly elevated in response to endoplasmic reticulum stressors and cytokines. These findings indicate that secretagogin is a potential novel biomarker, reflecting stress and islet cell dysfunction in T2D patients.