Isolated Rat Pancreatic Islets Research Articles

GIP_HUMAN [22-51] Peptide Encoded by the Glucose-Dependent Insulinotropic Polypeptide (GIP) Gene Suppresses Insulin Expression and Secretion in INS-1E Cells and Rat Pancreatic Islets.

GIP_HUMAN [22-51] is a recently discovered peptide that shares the same precursor molecule with glucose-dependent insulinotropic polypeptide (GIP). In vivo, chronic infusion of GIP_HUMAN [22-51] in ApoE-/- mice enhanced the development of aortic atherosclerotic lesions and upregulated inflammatory and proatherogenic proteins. In the present study, we evaluate the effects of GIP_HUMAN [22-51] on insulin mRNA expression and secretion in insulin-producing INS-1E cells and isolated rat pancreatic islets. Furthermore, we characterize the influence of GIP_HUMAN [22-51] on cell proliferation and death and on Nf-kB nuclear translocation. Rat insulin-producing INS-1E cells and pancreatic islets, isolated from male Wistar rats, were used in this study. Gene expression was evaluated using real-time PCR. Cell proliferation was studied using a BrdU incorporation assay. Cell death was quantified by evaluating histone-complexed DNA fragments. Insulin secretion was determined using an ELISA test. Nf-kB nuclear translocation was detected using immunofluorescence. GIP_HUMAN [22-51] suppressed insulin (Ins1 and Ins2) in INS-1E cells and pancreatic islets. Moreover, GIP_HUMAN [22-51] promoted the translocation of NF-κB from cytoplasm to the nucleus. In the presence of a pharmacological inhibitor of NF-κB, GIP_HUMAN [22-51] was unable to suppress Ins2 mRNA expression. Moreover, GIP_HUMAN [22-51] downregulated insulin secretion at low (2.8 mmol/L) but not high (16.7 mmol/L) glucose concentration. By contrast, GIP_HUMAN [22-51] failed to affect cell proliferation and apoptosis. We conclude that GIP_HUMAN [22-51] suppresses insulin expression and secretion in pancreatic β cells without affecting β cell proliferation or apoptosis. Notably, the effects of GIP_HUMAN [22-51] on insulin secretion are glucose-dependent.

Abstract #1401109: Quetiapine-induced Hypoglycemia in an Elderly Non-diabetic Patient with Dementia

Quetiapine is a second-generation antipsychotic agent (SGA) widely prescribed for the treatment of psychotic and mood disorders. In elderly patients, quetiapine is often used to treat psychotic symptoms associated with dementia. A metabolic syndrome phenotype of weight gain, hyperglycemia, and hyperlipidemia is a potential adverse effect of SGA’s, though hypoglycemia caused by SGA’s is documented. We report a case of quetiapine-induced hypoglycemia in a non-diabetic patient. A 79-year-old male with Lewy body dementia was admitted to hospital for management of a myocardial infarction. The patient had no history of diabetes mellitus, renal failure, or hepatic insufficiency, and nobody living with the patient was treated for diabetes. Plasma glucose on admission was 84 mg/dL. Quetiapine was started to control delirium, but within 24 h the patient had worsening confusion and a capillary blood glucose (CBG) measurement of 52 mg/dL. Symptoms improved with administration of dextrose, and the patient ultimately required a 10% dextrose infusion to maintain CBG’s >80 mg/dL. An ACTH-stimulation test (30 min stimulated cortisol 24.8 μg/dL, expected >18) and hypoglycemic medications panel (sulfonylureas and meglitinides) were unremarkable. Quetiapine was stopped, and hypoglycemia resolved. No hypoglycemia occurred during a subsequent 48-h diagnostic fast followed by a mixed meal tolerance test. Due to the patient’s advanced age and comorbidities, it was decided not re-challenge with quetiapine and observe for recurrence of hypoglycemia. Hypoglycemia is a rare complication of SGA’s, and diagnosis may be delayed or missed in patients with baseline confusion or agitation. In addition to quetiapine, there are case reports of hypoglycemia occurring during treatment with risperidone, olanzapine, aripiprazole, and paliperidone. Clozapine and olanzapine increase insulin release from isolated pancreatic islets in vitro, though quetiapine did not demonstrate this effect. However, endogenous hyperinsulinemia has been documented in case reports of quetiapine-induced hypoglycemia. The physiologic pathway by which SGA’s increase insulin release is unknown; some authors have focused on the antagonism of M1 muscarinic receptors, but the muscarinic M1 receptor antagonist pirenzepine has been demonstrated to reduce insulin secretion induced by the muscarinic receptor agonist carbachol from isolated rat pancreatic islets in vitro. Though the mechanism by which hypoglycemia occurs is unclear, this case demonstrates that hypoglycemia should be considered in patients with worsening mental status during treatment with SGA’s.

Submilligram Level of Beetle Antifreeze Proteins Minimize Cold-Induced Cell Swelling and Promote Cell Survival.

Hypothermic (cold) preservation is a limiting factor for successful cell and tissue transplantation where cell swelling (edema) usually develops, impairing cell function. University of Wisconsin (UW) solution, a standard cold preservation solution, contains effective components to suppress hypothermia-induced cell swelling. Antifreeze proteins (AFPs) found in many cold-adapted organisms can prevent cold injury of the organisms. Here, the effects of a beetle AFP from Dendroides canadensis (DAFP-1) on pancreatic β-cells preservation were first investigated. As low as 500 µg/mL, DAFP-1 significantly minimized INS-1 cell swelling and subsequent cell death during 4 °C preservation in UW solution for up to three days. However, such significant cytoprotection was not observed by an AFP from Tenebrio molitor (TmAFP), a structural homologue to DAFP-1 but lacking arginine, at the same levels. The cytoprotective effect of DAFP-1 was further validated with the primary β-cells in the isolated rat pancreatic islets in UW solution. The submilligram level supplement of DAFP-1 to UW solution significantly increased the islet mass recovery after three days of cold preservation followed by rewarming. The protective effects of DAFP-1 in UW solution were discussed at a molecular level. The results indicate the potential of DAFP-1 to enhance cell survival during extended cold preservation.

Novel Technology for Studying Insulin Secretion: Imaging and Quantitative Analysis by a Bioluminescence Method

We developed a method of video-rate bioluminescence imaging to visualize proteins secreted from living cells. A protein of interest was fused to Gaussia luciferase (GLase), and the luminescence signals of secreted GLase with coelenterazine (luciferin) were visualized at a video-rate of 30-500 ms/frame by using a water-cooled EM-CCD camera. We established a subclonal rat INS-1E cell line, named iGL cells, stably expressing the fusion protein of insulin and GLase (Insulin-GLase). By stimulation with high glucose, 3D-cultured iGL cells showed synchronized oscillatory secretion of insulin for over 1 h, as similarly observed in an isolated rat pancreatic islet. In 2D-cultured iGL cells, the luminescence images indicated that synchronized insulin secretion was localized in intercellular spaces between cells. Further, the relative amount of insulin secretion from iGL cells was easily determined with a luminometer, and we demonstrated that cell-cell interaction of beta cells is fundamental to increase glucose-stimulated insulin secretion by synchronization. Thus, iGL cells would be valuable for studying oscillatory insulin secretion and evaluating anti-diabetic drugs. Our bioluminescence imaging method with GLase could be generally used for investigating protein secretion in 2D and 3D cell culture systems.

FGF-1 modulates pancreatic β-cell functions/metabolism: An in vitro study

Fibroblast growth factor 1 (FGF-1), also known as acidic fibroblast growth factor (aFGF), is a growth factor and signaling protein encoded by the Fgf1 gene. Previous studies have shown that FGF-1 may also participate in the regulation of glucose metabolism, both in healthy organisms and in pathological conditions such as diabetes. Because insulin the main regulator of glucose metabolism is secreted from pancreatic beta cells, we investigated whether FGF-1 directly affects the secretion of this hormone and regulates the metabolism of beta cells and isolated pancreatic islets. By using insulin-producing INS-1E cells and isolated pancreatic islets, we investigated the effect of FGF-1 on cell proliferation, viability, apoptosis, and insulin expression and secretion. Our study showed that FGF1 and fibroblast growth factor receptors (FgfRs: FgfR1, FgfR2, FgfR3, and FgfR4) are present on mRNA level in INS-1E cells and isolated rat pancreatic islets. We also proved that FGF1 stimulates the proliferation of INS-1E beta cells and enhances the viability of these cells and that of isolated pancreatic islet cells, and that ERK1/2 kinase is involved in the regulation of INS-1E cell proliferation. Moreover, we found that FGF1 can stimulate insulin secretion from both INS-1E cells and isolated rat pancreatic islets. Thus, the FGF1 peptide increases cell survival and decreases cell death. The obtained results indicate that FGF1 may play a role in controlling the physiology and metabolism of pancreatic beta cells as well as glycemia.

Phoenixin-14 stimulates proliferation and insulin secretion in insulin producing INS-1E cells

Phoenixin (PNX) is a recently discovered neuropeptide which modulates appetite, pain sensation and neurons of the reproductive system in the central nervous system. PNX is also detectable in the circulation and in peripheral tissues. Recent data suggested that PNX blood levels positively correlate with body weight as well as nutritional status suggesting a potential role of this peptide in controlling energy homeostasis. PNX is detectable in endocrine pancreas, however it is unknown whether PNX regulates insulin biosynthesis or secretion. Using insulin producing INS-1E cells and isolated rat pancreatic islets we evaluated therefore, whether PNX controls insulin expression, secretion and cell proliferation. We identified PNX in pancreatic alpha as well as in beta cells. Secretion of PNX from pancreatic islets was stimulated by high glucose. PNX stimulated insulin mRNA expression in INS-1E cells. Furthermore, PNX enhanced glucose-stimulated insulin secretion in INS-1E cells and pancreatic islets in a time-dependent manner. Stimulation of insulin secretion by PNX was dependent upon cAMP/Epac signalling, while potentiation of cell growth and insulin mRNA expression was mediated via ERK1/2- and AKT-pathway. These results indicate that PNX may play a role in controlling glycemia by interacting with pancreatic beta cells.

Astragalin augments basal calcium influx and insulin secretion in rat pancreatic islets

Astragalin is a flavonol glycoside with several biological activities, including antidiabetic properties. The objective of this study was to investigate the effects of astragalin on glycaemia and insulin secretion, in vivo, and on calcium influx and insulin secretion in isolated rat pancreatic islets, ex vivo. Astragalin (1 and 10 mg / kg) was administered by oral gavage to fasted Wistar rats and serum glucose and plasma insulin were measured. Isolated pancreatic islets were used to measure basal insulin secretion and calcium influx. Astragalin (10 mg/ kg) decreased glycaemia and increased insulin secretion significantly at 15–180 min, respectively, in the glucose tolerance test. In isolated pancreatic cells, astragalin (100 μM) stimulated calcium influx through a mechanism involving ATP-dependent potassium channels, L-type voltage-dependent calcium channels, the sarcoendoplasmic reticulum calcium transport ATPase (SERCA), PKC and PKA. These findings highlight the dietary coadjuvant, astragalin, as a potential insulin secretagogue that may contribute to glucose homeostasis.

Supplementary cryoprotective effect of carboxylated ε-poly-l-lysine during vitrification of rat pancreatic islets

This study was designed to investigate whether cryosurvival of rat pancreatic islets can be improved by carboxylated ε-poly-l-lysine (CPLL). Islets isolated from Wistar × Brown-Norway F1 rats (101–200 μm in diameter) were cryopreserved in three vitrification solutions containing ethylene glycol (EG; 30%, v/v) and CPLL (0%, 10%, or 20%, v/v) by Cryotop® protocol (10 islets per device). The post-warm survival rate of the islets vitrified in the presence of 20% CPLL (74%), assessed by FDA/PI double staining, was higher than those in 0% and 10% CPLL (65% and 66%, respectively). Decreased EG concentrations (10% and 20%) in the presence of 20% CPLL resulted in impaired post-warm islet survival rates (50% and 64%, respectively). Value of stimulus index (SI) for 20 mM/3 mM glucose-stimulated insulin secretion was 4.1 in islets vitrified-warmed in the presence of 30% EG and 20% CPLL, which was comparable with those in fresh control islets and vitrified islets in 30% EG alone (4.1 and 4.4, respectively). A large number of islets (50 islets per device) could be cryopreserved in the presence of 30% EG and 20% CPLL by using nylon mesh as the device, without considerable loss of post-warm survival (68%) and SI value (3.7). In conclusion, supplementation of antifreeze 20% CPLL was effective in improving the post-warm survival of isolated rat pancreatic islets when vitrification solution containing 30% EG was used.

CAMP-PKA dependent ERK1/2 activation is necessary for vanillic acid potentiated glucose-stimulated insulin secretion in pancreatic β-cells

Vanillic acid (VA), a dietary phenolic compound is generally studied for its anti-oxidative and anti-inflammatory effects. However, the effect of VA on insulin secretion and its mechanism of action has never been explored. In this study, we report that VA augments glucose-stimulated insulin secretion (GSIS) in both insulin-secreting cell-line INS-1 and isolated rat pancreatic islets. Potentiation of GSIS is accompanied by a concurrent increase in 3′,5′-cyclic adenosine monophosphate (cAMP) and activation of protein kinase A (PKA) in INS-1 and rat islets. The activated cAMP-PKA pathway, in turn, phosphorylates extracellular signal-regulated kinases 1/2 (ERK1/2) in INS-1 cells. Pharmacological intervention with PKA and ERK1/2 inhibitors revealed that VA potentiated GSIS is primarily dependent on PKA mediated ERK1/2 activity. These findings demonstrated that VA directly acts on insulin-secreting pancreatic β-cells to exert its insulinotropic effect thereby providing a novel role of VA in the regulation of insulin secretion.

Assessment of Toxicity, Pro-Inflammatory Cytokines and Cytoprotective Potential of the Phytosaponin in Isolated Rat Pancreatic Islets

The objective of this study was to evaluate the in-depth toxicity assessment of pro-inflammatory and cytoprotective activity of phytosaponin. Saponin was isolated from methanolic extract of fruits of Momordica dioica and purification was achieved through the fractionation method of TLC. Toxicities were done as per OECD guidelines viz. In vivo micronucleus test 474 In vitro micronucleus test 487 In vitro chromosomal aberration test 473 in Neuroblastoma cells NB41A3 lines and Ames test 471. Saponin Momordica dioica was screened for production of pro-inflammatory cytokines viz. IL-1beta IL-6 and TNF-alpha was determined by ELISA against high glucose-induced cytoprotective activity like iNOS determined by western blot of against STZ-induced beta cell destruction in pancreatic isolated islets of rat. It was found that phytosaponin does not possess significant toxicities. Isolated pancreatic islet cells treated with high glucose produced pro-inflammatory cytokines which were reduced upon treatment with phytosaponin as compared to the high glucose control group. In similarly mediator of inflammation like iNOS was assessed and analysis showed SMD inhibited moderately in STZ stimulated pancreatic islet cells. The results concluded that phytosaponin possesses moderate to good cytoprotective nature and proves itself to be a potential candidate against diabetes

Investigation of β-Sitosterol and Prangol Extracted from Achillea Tenoifolia Along with Whole Root Extract on Isolated Rat Pancreatic Islets.

The genus Achillea (Asteraceae) consisting of important medicinal species, growing wildly in Iran, of which A. tenuifolia is found in Iran-o-Turan regions. Regarding the traditional use of Achillea species for treatment of diabetes and also lack of information on phyto-constituents of A. tenuifolia underground parts, in this study anti-diabetic activity of the plant have been reported. In order to find the main active components, underground parts of the plant were extracted with water and fractioned by hexane, ethyl acetate, and methanol and the separation of the main compounds were carried out via medium pressure liquid chromatography (MPLC). Also, anti-diabetic effects of the extract were investigated on rat pancreatic islets. The root extract of the plant as well as the compound β-sitosterol showed moderate α-amylase inhibitory activity, however prangol did not suppress the enzyme activity. The results of islet cells' bio-function assays revealed that the herb root extract was able to increase the secretion of insulin in high concentration (10 mg/mL) and improved the cell viability with no toxicity in all doses. Furthermore, the herbal extract could reduce the levels of reactive oxygen species (ROS) and lipid peroxidation (LPO). The plant extract also significantly decreased the enzyme activity for both caspase-3 and -9 and increased the antioxidant capacity of the isolated cells. Taking together, preparations or extracts from the underground parts of the plant are good candidates for further anti-diabetic investigation and clinical trials.

Morphological analysis of isolated rat pancreatic islets cultured under standard culture technique and with biopolymer microstructured collagen‑containing hydrogel

Introduction. Extracellular matrix play an essential role in providing structural integrity and physiological support to Langerhans islets in pancreas. Imitation of the native microenvironment can be useful for viability of isolated pancreatic islets in vitro and in vivo. Aim. The purpose of this study was to characterize and compare the effect of biopolymer microstructured collagen-containing hydrogel (BMCH) on isolated rat islets survival. Materials and methods. Islets were isolated by classic collagenase techniques with some modifications. There were used hystological, immunofluorescence and immunohystochemistry methods. Results. Rat islets cultured with collagen-based gel don’t revealed destructive changes of structure and remained viabile 7 days incubation. Conclusion. Positive effect of BMCH to rat islet survival was revealed.

Quantitative visualization of synchronized insulin secretion from 3D-cultured cells

Quantitative visualization of synchronized insulin secretion was performed in an isolated rat pancreatic islet and a spheroid of rat pancreatic beta cell line using a method of video-rate bioluminescence imaging. Video-rate images of insulin secretion from 3D-cultured cells were obtained by expressing the fusion protein of insulin and Gaussia luciferase (Insulin-GLase). A subclonal rat INS-1E cell line stably expressing Insulin-GLase, named iGL, was established and a cluster of iGL cells showed oscillatory insulin secretion that was completely synchronized in response to high glucose. Furthermore, we demonstrated the effect of an antidiabetic drug, glibenclamide, on synchronized insulin secretion from 2D- and 3D-cultured iGL cells. The amount of secreted Insulin-GLase from iGL cells was also determined by a luminometer. Thus, our bioluminescence imaging method could generally be used for investigating protein secretion from living 3D-cultured cells. In addition, iGL cell line would be valuable for evaluating antidiabetic drugs.

Unstable Expression of Commonly Used Reference Genes in Rat Pancreatic Islets Early after Isolation Affects Results of Gene Expression Studies.

The use of RT-qPCR provides a powerful tool for gene expression studies; however, the proper interpretation of the obtained data is crucially dependent on accurate normalization based on stable reference genes. Recently, strong evidence has been shown indicating that the expression of many commonly used reference genes may vary significantly due to diverse experimental conditions. The isolation of pancreatic islets is a complicated procedure which creates severe mechanical and metabolic stress leading possibly to cellular damage and alteration of gene expression. Despite of this, freshly isolated islets frequently serve as a control in various gene expression and intervention studies. The aim of our study was to determine expression of 16 candidate reference genes and one gene of interest (F3) in isolated rat pancreatic islets during short-term cultivation in order to find a suitable endogenous control for gene expression studies. We compared the expression stability of the most commonly used reference genes and evaluated the reliability of relative and absolute quantification using RT-qPCR during 0–120 hrs after isolation. In freshly isolated islets, the expression of all tested genes was markedly depressed and it increased several times throughout the first 48 hrs of cultivation. We observed significant variability among samples at 0 and 24 hrs but substantial stabilization from 48 hrs onwards. During the first 48 hrs, relative quantification failed to reflect the real changes in respective mRNA concentrations while in the interval 48–120 hrs, the relative expression generally paralleled the results determined by absolute quantification. Thus, our data call into question the suitability of relative quantification for gene expression analysis in pancreatic islets during the first 48 hrs of cultivation, as the results may be significantly affected by unstable expression of reference genes. However, this method could provide reliable information from 48 hrs onwards.

TRAF2 mediates JNK and STAT3 activation in response to IL-1β and IFNγ and facilitates apoptotic death of insulin-producing β-cells

Interleukin-1β (IL-1β) and interferon-γ (IFNγ) contribute to type 1 diabetes (T1D) by inducing β-cell death. Tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins are adaptors that transduce signaling from a variety of membrane receptors including cytokine receptors. We show here that IL-1β and IFNγ upregulate the expression of TRAF2 in insulin-producing INS-1E cells and isolated rat pancreatic islets. siRNA-mediated knockdown (KD) of TRAF2 in INS-1E cells reduced IL-1β-induced phosphorylation of JNK1/2, but not of p38 or ERK1/2 mitogen-activated protein kinases. TRAF2 KD did not modulate NFκB activation by cytokines, but reduced cytokine-induced inducible nitric oxide synthase (iNOS) promotor activity and expression. We further observed that IFNγ-stimulated phosphorylation of STAT3 required TRAF2. KD of TRAF2 or STAT3 reduced cytokine-induced caspase 3/7 activation, but, intriguingly, potentiated cytokine-mediated loss of plasma membrane integrity and augmented the number of propidium iodide-positive cells. Finally, we found that TRAF2 KD increased cytokine-induced production of reactive oxygen species (ROS). In summary, our data suggest that TRAF2 is an important mediator of IL-1β and IFNγ signaling in pancreatic β-cells.

Glucose-induced glutathione reduction in mitochondria is involved in the first phase of pancreatic β-cell insulin secretion

Glucose can acutely reduce mitochondrial glutathione redox state in rat islets. However, whether glucose-stimulated mitochondrial glutathione redox state relates to glucose-stimulated insulin secretion (GSIS) remains unknown. We used genetically encoded redox-sensitive GFPs to target the mitochondria to monitor glutathione redox changes during GSIS in rat pancreatic β-cells. The results showed that mitochondrial glutathione was more reduced during GSIS, whereas inhibition of this glutathione reduction impaired insulin secretion. In isolated rat pancreatic islets glutathione reduction in mitochondria and the first phase of GSIS were concurrence at the early stage of glucose-stimulation. Our results suggest that the glucose-induced glutathione reduction in mitochondria is primarily required for the first phase of GSIS.

Zinc oxide nanoparticles reduce apoptosis and oxidative stress values in isolated rat pancreatic islets.

Although toxic effects of zinc oxide nanoparticles (ZnO NPs) have been previously studied, there are still controversies in terms of dose, size, shape, and affecting cells. By such a perspective, in this study, small size of ZnO NPs with a diameter of 10 nm at low concentrations was studied for any effect on the viability and function of isolated rat pancreatic islets. Islets of Langerhans were isolated and assessed for viability, functionality (insulin secretion), cytosolic reactive oxygen species (ROS), and apoptosis by flow cytometry. The LC50 of ZnO NPs was found at 1,400 ng/mL at the first phase of the study. A meaningful increase in viability of islets and insulin secretion in basal and even stimulated concentrations of glucose was found by ZnO NPs (70 ng/mL) with p < 0.001 and p < 0.05, respectively. Likewise, ZnO NPs in 70 ng/mL concentration decreased cytosolic ROS generation (p < 0.05). In the meantime, the percentage of early stage of apoptotic cells dropped down to 17 % (from 29 % of control). These results for the first time confirm that ZnO NPs are not only safe when used at dose of 70 ng/mL but also improve viability and function of pancreatic islets and meanwhile reduce oxidative stress and prevent cells from entering the apoptotic phase.

Glucagon regulates orexin A secretion in humans and rodents.

Orexin A (OXA) modulates food intake, energy expenditure, and lipid and glucose metabolism. OXA regulates the secretion of insulin and glucagon, while glucose regulates OXA release. Here, we evaluate the role of glucagon in regulating OXA release both in vivo and in vitro. In a double-blind crossover study, healthy volunteers and type 1 diabetic patients received either intramuscular glucagon or placebo. Patients newly diagnosed with type 2 diabetes underwent hyperinsulinaemic-euglycaemic clamp experiments, and insulin-hypoglycaemia tests were performed on healthy volunteers. The primary endpoint was a change in OXA levels after intramuscular glucagon or placebo administration in healthy participants and patients with type 1 diabetes. Secondary endpoints included changes in OXA in healthy participants during insulin tolerance tests and in patients with type 2 diabetes under hyperinsulinaemic-euglycaemic conditions. Participants and staff conducting examinations and taking measurements were blinded to group assignment. OXA secretion in response to glucagon treatment was assessed in healthy and obese mice, the streptozotocin-induced mouse model of type 1 diabetes, and isolated rat pancreatic islets. Plasma OXA levels declined in lean volunteers and in type 1 diabetic patients injected with glucagon. OXA levels increased during hyperinsulinaemic hypoglycaemia testing in healthy volunteers and during hyperinsulinaemic euglycaemic conditions in type 2 diabetic patients. Plasma OXA concentrations in healthy lean and obese mice and in a mouse model of type 1 diabetes were lower after glucagon treatment, compared with vehicle control. Glucagon decreased OXA secretion from isolated rat pancreatic islets at both low and high glucose levels. OXA secretion declined in pancreatic islets exposed to diazoxide at high and low glucose levels, and after exposure to an anti-insulin antibody. Glucagon further reduced OXA secretion in islets pretreated with diazoxide or an anti-insulin antibody. Glucagon inhibits OXA secretion in humans and animals, irrespective of changes in glucose or insulin levels. Through modifying OXA secretion, glucagon may influence energy expenditure, body weight, food intake and glucose metabolism.

The insulin secretory action of novel polycyclic guanidines: Discovery through open innovation phenotypic screening, and exploration of structure–activity relationships

We report the discovery of the glucose-dependent insulin secretogogue activity of a novel class of polycyclic guanidines through phenotypic screening as part of the Lilly Open Innovation Drug Discovery platform. Three compounds from the University of California, Irvine, 1–3, having the 3-arylhexahydropyrrolo[1,2-c]pyrimidin-1-amine scaffold acted as insulin secretagogues under high, but not low, glucose conditions. Exploration of the structure–activity relationship around the scaffold demonstrated the key role of the guanidine moiety, as well as the importance of two lipophilic regions, and led to the identification of 9h, which stimulated insulin secretion in isolated rat pancreatic islets in a glucose-dependent manner.

O ‐GlcNAcylation of FoxO1 in pancreatic β cells promotes Akt inhibition through an IGFBP1‐mediated autocrine mechanism

O-GlcNAcylation on serine/threonine is a post-translational modification that controls the activity of nucleocytoplasmic proteins according to glucose availability. We previously showed that O-GlcNAcylation of FoxO1 in liver cells increases its transcriptional activity. In the present study, we evaluated the potential involvement of FoxO1 O-GlcNAcylation in the context of pancreatic β-cell glucotoxicity. FoxO1 was O-GlcNAcylated in INS-1 832/13 β cells and isolated rat pancreatic islets. O-GlcNAcylation of FoxO1 resulted in a 2-fold increase in its transcriptional activity toward a FoxO1 reporter gene and a 3-fold increase in the expression of the insulin-like growth factor-binding protein 1 (Igfbp1) gene at the mRNA level, resulting in IGFBP1 protein oversecretion by the cells. Of note, increased IGFBP1 in the culture medium inhibited the activity of the insulin-like growth factor 1 receptor (IGF1R)/phosphatidyl inositol 3 kinase (PI3K)/Akt pathway. We reveal in this report a novel mechanism by which O-GlcNAcylation inhibits Akt activity through an autocrine mechanism. However, although inhibition of IGFBP1 expression using siRNA restored the PI3 kinase/Akt pathway, it did not rescue INS-1 832/13 cells from high-glucose- or O-glcNAcylation-induced cell death. In contrast, FoxO1 down-regulation by siRNA led to 30 to 60% protection of INS-1 832/13 cells from death mediated by glucotoxic conditions. Therefore, whereas FoxO1 O-GlcNAcylation inhibits Akt through an IGFBP1-mediated autocrine pathway, the deleterious effects of FoxO1 O-GlcNAcylation on cell survival appeared to be independent of this pathway.