Diabetic Human Donors Research Articles

Ephrin-B2 PB-mononuclear cells reduce early post-stroke deficit in diabetic mice but not long-term memory impairment

Background and purposePost-stroke cognitive impairment (PSCI) has become a major public health issue, as a leading cause of dementia. The inflammation that develops soon after cerebral artery occlusion and may persist for weeks or months after stroke is a key component of PSCI. Our aim was to take advantage of the immunomodulatory properties of peripheral blood mononuclear cells (PB-MNC) stimulated with ephrin-B2/fc (PB-MNC+) for preventing PSCI. MethodsCortical infarct was induced by thermocoagulation of the middle cerebral artery in male diabetic mice (streptozotocin IP). PB-MNC were isolated from diabetic human donors, washed with recombinant ephrin-B2/Fc and injected into the mice intravenously on the following day. Infarct volume, sensorimotor deficit, cell death and immune cell densities were assessed on day 3. Six weeks later, cognitive assessment was performed using the Barnes maze. ResultsPB-MNC+ transplanted in post-stroke diabetic mice reduced the neurological deficit, infarct volume and apoptosis at D3, without modification of microglial cells, astrocytes and T-lymphocytes densities in the brain. Barnes maze assessment of memory showed that the learning, retention and reversal phases were not significantly modified by cell therapy. ConclusionsIntravenous PB-MNC+ administration the day after stroke induction in diabetic mice improved sensorimotor deficit and reduced infarct volume at the short term, but was unable to prevent long-term memory loss. To what extent diabetes impacts on cell therapy efficacy will have to be specifically investigated in the future. Including vascular risk factors systematically in preclinical studies of cell therapy will provide a comprehensive understanding of the mechanisms potentially limiting cell efficacy and also to identify good and bad responders, particularly in the long term.

The Human and Mouse Islet Peptidome: Effects of Obesity and Type 2 Diabetes, and Assessment of Intraislet Production of Glucagon-like Peptide-1.

To characterize the impact of metabolic disease on the peptidome of human and mouse pancreatic islets, LC-MS was used to analyze extracts of human and mouse islets, purified mouse alpha, beta, and delta cells, supernatants from mouse islet incubations, and plasma from patients with type 2 diabetes. Islets were obtained from healthy and type 2 diabetic human donors, and mice on chow or high fat diet. All major islet hormones were detected in lysed islets as well as numerous peptides from vesicular proteins including granins and processing enzymes. Glucose-dependent insulinotropic peptide (GIP) was not detectable. High fat diet modestly increased islet content of proinsulin-derived peptides in mice. Human diabetic islets contained increased content of proglucagon-derived peptides at the expense of insulin, but no evident prohormone processing defects. Diabetic plasma, however, contained increased ratios of proinsulin and des-31,32-proinsulin to insulin. Active GLP-1 was detectable in human and mouse islets but 100–1000-fold less abundant than glucagon. LC-MS offers advantages over antibody-based approaches for identifying exact peptide sequences, and revealed a shift toward islet insulin production in high fat fed mice, and toward proglucagon production in type 2 diabetes, with no evidence of systematic defective prohormone processing.

Leptin modulates pancreatic β-cell membrane potential through Src kinase–mediated phosphorylation of NMDA receptors

The adipocyte-derived hormone leptin increases trafficking of KATP and Kv2.1 channels to the pancreatic β-cell surface, resulting in membrane hyperpolarization and suppression of insulin secretion. We have previously shown that this effect of leptin is mediated by the NMDA subtype of glutamate receptors (NMDARs). It does so by potentiating NMDAR activity, thus enhancing Ca2+ influx and the ensuing downstream signaling events that drive channel trafficking to the cell surface. However, the molecular mechanism by which leptin potentiates NMDARs in β-cells remains unknown. Here, we report that leptin augments NMDAR function via Src kinase-mediated phosphorylation of the GluN2A subunit. Leptin-induced membrane hyperpolarization diminished upon pharmacological inhibition of GluN2A but not GluN2B, indicating involvement of GluN2A-containing NMDARs. GluN2A harbors tyrosine residues that, when phosphorylated by Src family kinases, potentiate NMDAR activity. We found that leptin increases phosphorylation of Tyr-418 in Src, an indicator of kinase activation. Pharmacological inhibition of Src or overexpression of a kinase-dead Src mutant prevented the effect of leptin, whereas a Src kinase activator peptide mimicked it. Using mutant GluN2A overexpression, we show that Tyr-1292 and Tyr-1387 but not Tyr-1325 are responsible for the effect of leptin. Importantly, β-cells from db/db mice, a type 2 diabetes mouse model lacking functional leptin receptors, or from obese diabetic human donors failed to respond to leptin but hyperpolarized in response to NMDA. Our study reveals a signaling pathway wherein leptin modulates NMDARs via Src to regulate β-cell excitability and suggests NMDARs as a potential target to overcome leptin resistance.

SUN-654 Dynamic and Regional Variation of Pancreatic Innervation in Diabetes

Background: The pancreas is a highly heterogeneous organ, with regional anatomical, developmental and functional differences. The endocrine pancreas is densely innervated, and neural signals play a significant role in glucose regulation by modulating pancreatic hormone release. However, relatively little is known about the anatomical relationships between islets and nerves across the whole pancreas. Since thin filamentous structures, such as nerves, are difficult to quantify and trace over large volumes using thin section histology, there is a need for high resolution imaging and rendering of intact pancreatic tissue in 3D. Aim: To use optical clearing, whole organ imaging, and 3D rendering to quantify islets and innervation across the whole pancreas in healthy mice, in two mouse models of diabetes, and in pancreatic samples from nondiabetic and diabetic human donors. Methods: Whole-mount staining and clearing was performed using iDISCO+ to quantify innervation, defined by the neuronal marker NF200, and beta cells in pancreata from C57Bl/6 mice, non-obese diabetic (NOD) mice, streptozotocin (STZ)-treated mice, and in pancreatic samples from nondiabetic and diabetic human donors. Z-stacked optical sections were acquired with an Ultramicroscope II at 4x or 12x magnification. Imaris was used to create digital surfaces covering the NF200+ innervation and islets to automatically determine innervation density and islet/nerve interactions. Results: Beta cell volumes were 1-4% in the human pancreas, and 1-2% in the healthy mouse pancreas, with regional variations in islet volume and insulin intensity. There were also significant differences in islet biology between the diabetes models. Innervation of the endocrine pancreas was significantly enriched compared to the surrounding exocrine pancreas, with regional variation. Islets were closely associated with pancreatic innervation and decreased in size with increasing distance from nerves in both mouse and human pancreatic tissue. Innervated islets were relatively preserved in models of diabetes. Finally, islet innervation and expression of neural markers were higher in human samples from diabetic patients and in mouse models of diabetes, with temporal and regional differences. Conclusions: 3D imaging and unbiased analysis across the whole pancreas provides comprehensive measurement of pancreatic nerve volumes and distribution. It allows detailed analysis of the anatomical relationship between nerves and islets, and reveals a close association that is maintained across species. The relative enrichment of innervated islets in diabetes and dynamic changes in islet innervation during the development of diabetes suggest further work is needed to examine the role of pancreatic nerves in preserving and protecting beta cells.

MiRNAs responsive to the diabetic microenvironment in the human beta cell line EndoC-βH1 may target genes in the FOXO, HIPPO and Lysine degradation pathways

Altered expression of miRNAs is evident in the islets of diabetic human donors, but the effects of specific aspects of the diabetic microenvironment and identity of gene ontology pathways demonstrating target gene enrichment in response to each is understudied.We assessed changes in the miRNA milieu in response to high/low glucose, hypoxia, dyslipidaemia and inflammatory factors in a humanised EndoC-βH1 beta cell culture system and performed miRPath analysis for each treatment individually. The 10 miRNAs demonstrating the greatest dysregulation across treatments were then independently validated and Gene Set Enrichment Analysis to confirm targeted pathways undertaken.171 of 392 miRNAs displayed altered expression in response to one or more cellular stressors. miRNA changes were treatment specific, but their target genes were enriched in conserved pathways. 5 miRNAs (miR-136-5p, miR299-5p, miR-454-5p, miR-152 and miR-185) were dysregulated in response to multiple stressors and survived validation in independent samples (p = 0.008, 0.002, 0.012, 0.005 and 0.024 respectively). Target genes of dysregulated miRNAs were clustered into FOXO1, HIPPO and Lysine degradation pathways (p = 0.02, p = 5.84 × 10−5 and p = 3.00 × 10−3 respectively).We provide evidence that the diabetic microenvironment may induce changes to the expression of miRNAs targeting genes enriched in pathways involved in cell stress response and cell survival.

SAT-LB035 The G Protein-Coupled Estrogen Receptor GPER Plays a Dominant Role in Human Islet Cell Proliferation upon High Glucose

Estrogen exerts its effects through genomic and rapid extra-nuclear signaling pathways. The classic estrogen receptors, ERα and ERβ have demonstrated favorable effects on beta-cell function and survival but their clinical utility has been questioned owing to oncogenic potential and concerns regarding feminizing effects in men. Recent work indicates, however, that the G-protein coupled estrogen receptor (GPER), expressed in pancreatic islet cells retains the estrogenic beta-cell protective effects, without the shortcomings of the classic ERs. GPER activation enhances glucose stimulated insulin secretion, reduces islet apoptosis upon insult and has been implicated in beta-cell mass expansion during pregnancy in rodent islets. Since GPER can thus comprise an attractive treatment target in hyperglycemic states we examined the effects of the three ERs on proliferation of human pancreatic islets in the presence of hyperglycemia. Islets from eight non diabetic human donors (6 males and 2 females) were grown at glucose concentrations of either 11mM or 25mM for 24 hours and examined for DNA synthesis after treatment with E2 (at increasing concentrations) and with specific agonists for ERα, ERβ and GPER (PPT 10nM, DPN 10nM and G1 100nM, respectively). At a glucose concentration of 11mM, E2 and all three ER agonists induced a significant ~2 folds increase in 3[H]-thymidine incorporation. However, under still higher glucose conditions (HG; 25mM) only the GPER agonist G1 increased proliferation equipotently (~2.5 folds; p<0.001), while stimulation of ERα and ERβ enhanced proliferation by ~1.4 folds only (p<0.05 for ERα). In search for an explanation for the differential effects, we examined expression of the three ERs at glucose concentrations of 11mM and 25mM, by qRT-PCR. High glucose reduced expression of ERα and ERβ (by 15%; p<0.05) but not the expression of GPER. Although beta-cells comprise the majority (~70%) of cells in human islets, expression of GPER has also been demonstrated in alpha and delta cells and thus the full effect of hyperglycemia on GPER expression in beta-cells may be masked. Hence, extending previous reports, our results suggest that GPER may comprise an attractive target in the therapy of human diabetes and point to the phenomenon of species specificity regarding effects of glucose on estrogen receptor subtype expression and activity. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Diabetes relief in mice by glucose-sensing insulin-secreting human α-cells.

Cell identity switches, where terminally-differentiated cells convert into different cell-types when stressed, represent a widespread regenerative strategy in animals, yet they are poorly documented in mammals. In mice, some glucagon-producing pancreatic α-cells and somatostatin-producing δ-cells become insulin expressers upon ablation of insulin-secreting β-cells, promoting diabetes recovery. Whether human islets also display this plasticity, especially in diabetic conditions, remains unknown. Here we show that islet non-β-cells, namely α-cells and PPY-producing γ–cells, obtained from deceased non-diabetic or diabetic human donors, can be lineage-traced and reprogrammed by the transcription factors Pdx1 and MafA to produce and secrete insulin in response to glucose. When transplanted into diabetic mice, converted human α-cells reverse diabetes and remain producing insulin even after 6 months. Surprisingly, insulin-producing α-cells maintain α-cell markers, as seen by deep transcriptomic and proteomic characterization. These observations provide conceptual evidence and a molecular framework for a mechanistic understanding of in situ cell plasticity as a treatment for diabetes and other degenerative diseases.

338 - Mitochondrial DNA damage in diabetic retinopathy: role of epigenetics

Purpose Diabetes dysfunctions retinal mitochondria and increases base pair mismatches in mitochondrial DNA (mtDNA), and damaged mtDNA impairs the electron transport chain system. A mismatch repair system actively repairs the uncomplimentary base pairs, but in diabetes, the enzyme responsible for cutting the base-mismatches, MutL homolog 1 (Mlh1), is also compromised, which further adds to the damage of mtDNA. In diabetes, retinal DNA methylation machinery is also altered, and the activity of DNA methyl transferases (Dnmts) is significantly increased. Since Mlh1 promoter has over 50 CpG sites, making it a good target for DNA methylation, our aim was to investigate the role of DNA methylation in mtDNA damage in the development of diabetic retinopathy. Methods Retinal microvessels from streptozotocin-induced diabetic mice, with or without Dnmt inhibitor 5-azacytidine (Aza, 2.5mg/kg, intraperitoneal), were analyzed for methylated cytosine (5mC) levels using immuno-capture method and Mlh1 promoter- specific primers. Base-mismatch was quantified by digesting mtDNA amplicons using mismatch specific surveyor endonuclease, followed by DNA fragmentation analysis. Similar experiments were performed in retinal microvessels prepared from human donors with documented diabetic retinopathy. Results Compared to normal mice, retinal microvessels from diabetic mice had decreased Mlh1 expression and increased Dnmt1 and 5mC levels at Mlh1 promoter. In the same mice, base-mismatches in their mtDNA were also increased. Administration of Aza ameliorated diabetes-induced decrease in Mlh1 expression, and prevented increase in Dnmt1 and DNA methylation of Mlh1 promoter. Similarly, compared to age-matched non diabetic human donors, retinal microvessels from donors with established diabetic retinopathy had decreased levels of Mlh1 and increased levels of Dnmt1 and 5mC at Mlh1 promoter. Conclusions Thus, DNA methylation of Mlh1 promoter has a major role in impaired mtDNA damage. Therapies targeted to halt DNA methylation could have potential to prevent/halt mtDNA damage, and the development of diabetic retinopathy.

Statistical Frailty Modeling for Quantitative Analysis of Exocytotic Events Recorded by Live Cell Imaging: Rapid Release of Insulin-Containing Granules Is Impaired in Human Diabetic β-cells.

Hormones and neurotransmitters are released when secretory granules or synaptic vesicles fuse with the cell membrane, a process denoted exocytosis. Modern imaging techniques, in particular total internal reflection fluorescence (TIRF) microscopy, allow the investigator to monitor secretory granules at the plasma membrane before and when they undergo exocytosis. However, rigorous statistical approaches for temporal analysis of such exocytosis data are still lacking. We propose here that statistical methods from time-to-event (also known as survival) analysis are well suited for the problem. These methods are typically used in clinical settings when individuals are followed over time to the occurrence of an event such as death, remission or conception. We model the rate of exocytosis in response to pulses of stimuli in insulin-secreting pancreatic β-cell from healthy and diabetic human donors using piecewise-constant hazard modeling. To study heterogeneity in the granule population we exploit frailty modeling, which describe unobserved differences in the propensity to exocytosis. In particular, we insert a discrete frailty in our statistical model to account for the higher rate of exocytosis in an immediately releasable pool (IRP) of insulin-containing granules. Estimates of parameters are obtained from maximum-likelihood methods. Since granules within the same cell are correlated, i.e., the data are clustered, a modified likelihood function is used for log-likelihood ratio tests in order to perform valid inference. Our approach allows us for example to estimate the size of the IRP in the cells, and we find that the IRP is deficient in diabetic cells. This novel application of time-to-event analysis and frailty modeling should be useful also for the study of other well-defined temporal events at the cellular level.

Effects of activin A on survival, function and gene expression of pancreatic islets from non-diabetic and diabetic human donors

Emerging evidence suggests that activin with its associated receptors, second messengers, and antagonists would be excellent targets for therapeutic drug development in the treatment of diabetes. We undertook the current study to investigate the ability to extrapolate findings from rodent studies to human islets in which data thus far has been scarce. We tested the hypothesis that human islets synthesize activin and that activin participates in the regulation of islet β-cells. Human islets from 33 separate isolations were categorized based on functional status, culture status and diabetic status. Statistical comparisons were made by ANOVA with Tukey post-hoc adjustment for multiple comparisons. Experiments investigating activin utilized qPCR, FACS cell sorting, immunofluorescent antibody staining, functionality assays, viability assays and protein secretion assays. We have defined the transcript expression patterns of activin and the TGFβ superfamily in human islets. We found INHBA (the gene encoding activin A) to be the most highly expressed of the superfamily in normal, cultured islets. We elucidated a link between the islet microenvironment and activin A. We found differential ligand expression based on diabetic, culture and functional status. Further, this is also the first report that links direct effects of activin A with the ability to restore glucose-stimulated insulin secretion in human islets from type 2 diabetic donors thereby establishing the relevance of targeting activin for therapeutic drug development.

DNA methylation of the glucagon-like peptide 1 receptor (GLP1R) in human pancreatic islets

BackgroundInsulin secretion is enhanced upon the binding of Glucagon-like peptide-1 (GLP-1) to its receptor (GLP1R) in pancreatic β cells. Although a reduced expression of GLP1R in pancreatic islets from type 2 diabetic patients and hyperglycaemic rats has been established, it is still unknown if this is caused by differential DNA methylation of GLP1R in pancreatic islets of type 2 diabetic patients.MethodsIn this study, DNA methylation levels of 12 CpG sites close to the transcription start site of GLP1R were analysed in pancreatic islets from 55 non-diabetic and 10 type 2 diabetic human donors as well as in β and α cells isolated from human pancreatic islets. DNA methylation of GLP1R was related to GLP1R expression, HbA1c levels and BMI. Moreover, mRNA expression of MECP2, DNMT1, DNMT3A and DNMT3B was analysed in pancreatic islets of the non-diabetic and type 2 diabetic donors.ResultsOne CpG unit, at position +199 and +205 bp from the transcription start site, showed a small increase in DNA methylation in islets from donors with type 2 diabetes compared to non-diabetic donors (0.53%, p=0.02). Furthermore, DNA methylation levels of one CpG site located 376 bp upstream of the transcription start site of GLP1R correlated negatively with GLP1R expression (rho=−0.34, p=0.008) but positively with BMI and HbA1c (rho=0.30, p=0.02 and rho=0.30, p=0.03, respectively). This specific CpG site is located in an area with known SP1 and SP3 transcription factor binding sites. Moreover, when we compared the DNA methylation of the GLP1R promoter in isolated human β and α cells, we found that it was higher in α- compared with β-cells (p=0.009). Finally, there was a trend towards decreased DNMT3A expression (p=0.056) in type 2 diabetic compared with non-diabetic islets.ConclusionsTogether, our study shows that while BMI and HbA1c are positively associated with DNA methylation levels of GLP1R, its expression is negatively associated with DNA methylation of GLP1R in human pancreatic islets.

IGF-I mRNA and Signaling in the Diabetic Retina

IGF-I promotes the survival of multiple cell types by activating the IGF-I receptor (IGF-IR), which signals downstream to a serine/threonine kinase termed Akt. Because in diabetes vascular and neural cells of the retina undergo accelerated apoptosis, we examined IGF-I synthesis and signaling in the human and rat diabetic retina. In retinas obtained postmortem from six donors aged 64 +/- 8 years with a diabetes duration of 7 +/- 5 years, IGF-I mRNA levels were threefold lower than in the retinas of six age-matched nondiabetic donors (P = 0.005). In the retinas of rats with 2 months' duration of streptozotocin-induced diabetes, IGF-I mRNA levels were similar to those of control rats, but after 5 months of diabetes they failed to increase to the levels recorded in age-matched controls (P < 0.02). Retinal IGF-I expression was not altered by hypophysectomy, proving to be growth-hormone independent. IGF-IR levels were modestly increased in the human diabetic retinas (P = 0.02 vs. nondiabetic retinas) and were unchanged in the diabetic rats. Phosphorylation of the IGF-IR could be measured only in the rat retina, and was not decreased in the diabetic rats (94 +/- 18% of control values). In the same diabetic rats, phosphorylation of Akt was 123 +/- 21% of control values. There was not yet evidence of increased apoptosis of retinal microvascular cells after 5 months of streptozotocin-induced diabetes. Hence, in the retina of diabetic rats, as in the retina of diabetic human donors, IGF-I mRNA levels are substantially lower than in age-matched nondiabetic controls, whereas IGF-IR activation and signaling are not affected, at least for some time. This finding suggests that in the diabetic retina, the activation of the IGF-IR is modulated by influences that compensate for, or are compensated by, decreased IGF-I synthesis.