Rat Islets Research Articles

Tip 2 Diyabetik Sıçanlarda Kurkuminin Streptozosin Kaynaklı Pankreatik Adacık Hasarına Karşı Koruyucu Etkisi: Deneysel Bir Çalışma

Objective: The aim of study of was to determine the damage caused by streptozocin (STZ)-induced Type 2 diabetes in pancreatic islet in rats and the effect of curcumin on this damage. Material and Methods: A total of 32 (250-320 g) Wistar albino male rats were used. Rats were randomly divided into 4 groups as Control (C), Diabetes (DM), Curcumin (CUR), and Diabetes+Curcumin (DM+CUR) (n=8). Histopathological, stereological and immunohistochemical analyzes were used to determine the damage caused by type 2 diabetes in the pancreatic tissue and the effect of curcumin on this damage. Results: In the histopathological evaluation of pancreatic tissues, in pancreatic sections of DM group rats, the islets were smaller and their borders were irregular, and degenerative and necrotic changes were observed in the cells. In the DM+CUR group, it was determined that these degenerative changes in the islets of Langerhans and cells were alleviated. In the stereological analysis, it was determined that the islet volume, the ratio of islets to the pancreas and the number of beta cells in the DM group were statistically significantly decreased compared to the other groups, and it was found to be higher in the DM+CUR group than in the DM group (p<0.05). In the immunohistochemical evaluation, the C and CUR groups showed similar staining with Ki67, whereas the staining was less in the diabetic group. In the diabetes group treated with curcumin, it was found that curcumin attenuated the diabetes- related Ki67 reduction. Conclusion: In this study, it was determined that curcumin reduced the damage in the islets and beta cells in the pancreas of rats with diabetes mellitus with STZ and provided a significant protection in the pancreatic tissue.

Neu-P11 - a novel melatonin receptor agonist, could improve the features of type-2 diabetes mellitus in rats.

Melatonin (Mel) and its receptors are promising for glycaemic control in patients with type 2 diabetes mellitus (T2DM) and its complications, but there is significant heterogeneity among studies. This study aims to investigate the effects of Mel receptor agonist Neu-P11 on glucose metabolism, immunity, and islet function in T2DM rats. In this study, SD rats were treated with a high-fat diet and streptozotocin (STZ) to establish a T2DM model. The glucose oxidase method was used to measure blood glucose levels. Glucose and insulin tolerance tests were used to assess glucose metabolism. Haematoxylin-eosin staining was used to observe pancreatic tissue injury. The apoptosis of isletβ cells was analysed by TUNEL and insulin staining. Reactive oxygen species (ROS) levels and immune cell expression were analysed by flow cytometry. IF was used to analyse the activation of microglia. The immunoglobulins: IgA, IgG, IgM, tumour necrosis factorα (TNF-α), interleukins IL-10 and IL-1β, interferonγ (IFN-γ), C-peptide, and insulin levels were determined by ELISA. The expression of CD11b, CD86, cleaved caspase 3, p21, and P16 proteins were analysed by western blot. The results showed that the blood glucose level increased, insulin resistance occurred, spleen coefficient and ROS levels increased, humoral immunity in peripheral blood decreased, and inflammation increased in the model group compared to the control group. After Mel and Neu-P11 treatment, the blood glucose level decreased significantly, insulin sensitivity improved, spleen coefficient and ROS levels decreased, humoral immunity in peripheral blood was enhanced, and inflammation improved in T2DM rats. Brain functional analysis of T2DM rats showed that microglia cells were activated, TNF-α and IL-β levels were increased, and IL-10 levels were decreased. Mel and Neu-P11 treatment reversed these indexes. Functional analysis of islets in T2DM rats showed that islet structure inflammation was impaired, isletβ cells were apoptotic, p21 and p16 protein expressions were increased, and blood C-peptide and insulin were decreased. Mel and Neu-P11 treatment restored the function of pancreatic b cells and improved the damage of pancreatic tissue. Melatonin and its receptor Neu-P11 can reduce the blood glucose level, enhance humoral and cellular immunity, inhibit microglia activation and inflammation, and repair isletβ cell function, and this improve the characterization of T2DM-related diseases.

The Protective impact of lycopene and folic acid supplementation against nicotine toxicity on pancreatic islets in adult male albino rats: Biochemical &amp;immuno-histochemical study

The Protective impact of lycopene and folic acid supplementation against nicotine toxicity on pancreatic islets in adult male albino rats: Biochemical &amp;immuno-histochemical study

Gut Metabolite Trimethylamine N-Oxide Protects INS-1 β-Cell and Rat Islet Function under Diabetic Glucolipotoxic Conditions.

Serum accumulation of the gut microbial metabolite trimethylamine N-oxide (TMAO) is associated with high caloric intake and type 2 diabetes (T2D). Impaired pancreatic β-cell function is a hallmark of diet-induced T2D, which is linked to hyperglycemia and hyperlipidemia. While TMAO production via the gut microbiome-liver axis is well defined, its molecular effects on metabolic tissues are unclear, since studies in various tissues show deleterious and beneficial TMAO effects. We investigated the molecular effects of TMAO on functional β-cell mass. We hypothesized that TMAO may damage functional β-cell mass by inhibiting β-cell viability, survival, proliferation, or function to promote T2D pathogenesis. We treated INS-1 832/13 β-cells and primary rat islets with physiological TMAO concentrations and compared functional β-cell mass under healthy standard cell culture (SCC) and T2D-like glucolipotoxic (GLT) conditions. GLT significantly impeded β-cell mass and function by inducing oxidative and endoplasmic reticulum (ER) stress. TMAO normalized GLT-mediated damage in β-cells and primary islet function. Acute 40µM TMAO recovered insulin production, insulin granule formation, and insulin secretion by upregulating the IRE1α unfolded protein response to GLT-induced ER and oxidative stress. These novel results demonstrate that TMAO protects β-cell function and suggest that TMAO may play a beneficial molecular role in diet-induced T2D conditions.

A Safe, Fibrosis-Mitigating, and Scalable Encapsulation Device Supports Long-Term Function of Insulin-Producing Cells.

Encapsulation and transplantation of insulin-producing cells offer a promising curative treatment for type 1 diabetes (T1D) without immunosuppression. However, biomaterials used to encapsulate cells often elicit foreign body responses, leading to cellular overgrowth and deposition of fibrotic tissue, which in turn diminishes mass transfer to and from transplanted cells. Meanwhile, the encapsulation device must be safe, scalable, and ideally retrievable to meet clinical requirements. Here, a durable and safe nanofibrous device coated with a thin and uniform, fibrosis-mitigating, zwitterionically modified alginate hydrogel for encapsulation of islets and stem cell-derived beta (SC-β) cells is reported. The device with a configuration that has cells encapsulated within the cylindrical wall, allowing scale-up in both radial and longitudinal directions without sacrificing mass transfer, is designed. Due to its facile mass transfer and low level of fibrotic reactions, the device supports long-term cell engraftment, correcting diabetes in C57BL6/J mice with rat islets for up to 399 days and SCID-beige mice with human SC-β cells for up to 238 days. The scalability and retrievability in dogs are further demonstrated. These results suggest the potential of this new device for cell therapies to treat T1D and other diseases.

Identification of Novel Regulatory Regions Induced by Intrauterine Growth Restriction in Rat Islets.

Intrauterine growth restriction (IUGR) leads to the development of type 2 diabetes in adulthood, and the permanent alterations in gene expression implicate an epigenetic mechanism. Using a rat model of IUGR, we performed TrueSeq-HELP Tagging to assess the association of DNA methylation changes and gene dysregulation in islets. We identified 511 differentially methylated regions (DMRs) and 4377 significantly altered single CpG sites. Integrating the methylome and our published transcriptome data sets resulted in the identification of pathways critical for islet function. The identified DMRs were enriched with transcription factor binding motifs, such as Elk1, Etv1, Foxa1, Foxa2, Pax7, Stat3, Hnf1, and AR. In silico analysis of 3-dimensional chromosomal interactions using human pancreas and islet Hi-C data sets identified interactions between 14 highly conserved DMRs and 35 genes with significant expression changes at an early age, many of which persisted in adult islets. In adult islets, there were far more interactions between DMRs and genes with significant expression changes identified with Hi-C, and most of them were critical to islet metabolism and insulin secretion. The methylome was integrated with our published genome-wide histone modification data sets from IUGR islets, resulting in further characterization of important regulatory regions of the genome altered by IUGR containing both significant changes in DNA methylation and specific histone marks. We identified novel regulatory regions in islets after exposure to IUGR, suggesting that epigenetic changes at key transcription factor binding motifs and other gene regulatory regions may contribute to gene dysregulation and an abnormal islet phenotype in IUGR rats.

Intra-islet glucagon confers β-cell glucose competence for first-phase insulin secretion and favors GLP-1R stimulation by exogenous glucagon

We report that intra-islet glucagon secreted from α-cells signals through β-cell glucagon and GLP-1 receptors (GcgR and GLP-1R), thereby conferring to rat islets their competence to exhibit first-phase glucose-stimulated insulin secretion (GSIS). Thus, in islets not treated with exogenous glucagon or GLP-1, first-phase GSIS is abolished by a GcgR antagonist (LY2786890) or a GLP-1R antagonist (Ex[9–39]). Mechanistically, glucose competence in response to intra-islet glucagon is conditional on β-cell cAMP signaling because it is blocked by the cAMP antagonist prodrug Rp-8-Br-cAMPS-pAB. In its role as a paracrine hormone, intra-islet glucagon binds with high affinity to the GcgR, while also exerting a “spillover” effect to bind with low affinity to the GLP-1R. This produces a right shift of the concentration-response relationship for the potentiation of GSIS by exogenous glucagon. Thus, 0.3 nM glucagon fails to potentiate GSIS, as expected if similar concentrations of intra-islet glucagon already occupy the GcgR. However, 10 to 30 nM glucagon effectively engages the β-cell GLP-1R to potentiate GSIS, an action blocked by Ex[9–39] but not LY2786890. Finally, we report that the action of intra-islet glucagon to support insulin secretion requires a step-wise increase of glucose concentration to trigger first-phase GSIS. It is not measurable when GSIS is stimulated by a gradient of increasing glucose concentrations, as occurs during an oral glucose tolerance test in vivo. Collectively, such findings are understandable if defective intra-islet glucagon action contributes to the characteristic loss of first-phase GSIS in an intravenous glucose tolerance test, that is, diagnostic of type 2 diabetes in the clinical setting.

402.7: Homemade Hydrogel From Human Amniotic Membrane Improves Islet Transplantation Outcomes in Diabetic Immunodeficient Mice

Background: Neovascularized devices and biopolymer scaffolds are getting a great attention for their potential to improve islet transplantations. Among many biomaterials sources, human amniotic membrane (HAM) is considered as a natural source to produce ECM-based hydrogels with immunomodulatory, anti-inflammatory and antifibrotic properties. Here we develop an ECM-based hydrogel (Amniogel) derived from HAM and evaluate its potential to support islet function in vitro and in vivo. Methods: The Amniogels were generated from HAM and assessed for porosity, for ECM content and fibre integrity. To assess Amniogel impact on islet viability and function, isolated rat islets were incorporated into the Amniogel and cultured for one week. The cell viability was evaluated and islet function was assessed by a glucose stimulated insulin secretion (GSIS) test. We assessed whether incorporation of islets into the Amniogel could enhance engraftment and lead to better glycaemic control in diabetic Nod-Rag mice. For this purpose, 250 rat islets (IEQ) loaded into the Amniogel or islets alone (control) were transplanted into the epididymal fat of diabetic NSG mice. Blood glucose levels were monitored daily and intraperitoneal glucose tolerance tests were carried out. Grafts and serum were harvested at 1, 2, 6 and 12 weeks to assess outcome. Results: The ECM concentration in the Amniogel affected the pore size. Insulin expression and viability of islets incorporated into Amniogel was significantly higher than inn the control group. In addition, significant enhancement of GSIS was observed from islets embedded in Amniogel. In vivo experiments showed that transplantation of 250 IEQ embedded in Amniogel lead to enhanced engraftment, vascularization, viability and better glycaemic control compared to control mice. Conclusions: Incorporation of pancreatic islet into amnion-derived Amniogel enhances islet engraftment and is a valuable approach to improve islet transplantation outcomes.

P.122: Development of an Oxygen Durability Model to Overcome Hypoxia in Encapsulated Islets Within a Functionalized Oxygenated Biomaterial

Introduction: Post-transplantation islet survival is inherently limited by the lack of suitable support matrix and insufficient oxygen supply (hypoxia), which is aggravated in macroencapsulation devices.Graft failure can be overcome by cell encapsulation in highly oxygenated functionalized biomaterials able to provide oxygen and extracellular matrix (ECM) support. An estimation of the oxygen durability in these encapsulation matrices is critical to define the cell density and implant size for optimal graft survival, especially until the macroencapsulation device is sufficiently re-vascularized. The aim of this study was to develop a predictive mathematical model that could estimate the oxygen lifetime of an oxygenated biomaterial (Oxygel) encapsulating different diabetes-relevant cell lines. Methods: Oxygel was formulated by the shear mixing of a physically crosslinked native Hyaluronic acid hydrogel and a Perfluocarbon/lipoid nanoemulsion. Oxygel was oxygenated by flushing oxygen using a customized static Y-mixer while the gel was infused with a syringe pump or using a designed scaled-up oxygenator. Post-oxygenation O2 levels of Oxygel and controls were monitored by microinvasive fibre optic needle-type oxygen microsensors (n=3). Oxygen diffusion coefficient (Dv) was estimated by the experimental fitting of the oxygen release profiles to a diffusion model governed by Fick’s second law. Oxygen durability predictions were performed using a non-diffusion-controlled model considering: (i) recorded initial oxygen tension, (ii) gel concentration and Ostwald oxygen solubility constants of different gel components, (iii) cell density and (iv) oxygen consumption rate (OCR) of the studied cell line. Different diabetes-relevant cell lines (INS-1E, iPS derived B-cells, rat islets, human islets) were modelled using experimental (Agilent Seahorse analyzer) and/or bibliographic OCR values. Summary of results: A self-stable and shear-thinning gel was successfully formulated and oxygenated. A high initial oxygen tension was achieved (650–700 torr, 87–94 % O2 saturation), that was diminished after the cell encapsulation/dilution step (520–550 torr, 70–74 % O2 saturation, Fig 1). Acellular oxygel released oxygen for 100 h until stabilization at atmospheric levels, presenting a 14-times smaller Dv in Oxygel than in PBS. The impact of the model parameters in cell lines with different OCR values was investigated, showing shorter oxygen lifetime in islets (or IEQ) compared to single cell analogues.Conclusion: A highly oxygenated encapsulation matrix for enhanced islet survival was developed and their properties as oxygen carrier evaluated, showing a high oxygen payload and prolonged sustained release properties. Model predictions shows the importance of islet O2 metabolism in the design of macroencapsulation devices and supporting ECM. G.P.D. and D.A.D.-L. acknowledge the DELIVER project that received funding from the European Union’s Horizon 2020 Marie Skłodowska-Curie Actions programme under grant agreement number 812865. G.P.D. acknowledges funding from Science Foundation Ireland under grant SFI/12/RC/2278, Advanced Materials and Bioengineering Research (AMBER) Centre and financial support from the College of Medicine, Nursing and Health Sciences, National University of Ireland Galway.

P.169: Decellularized Cotyledons Isolated From Human Placenta Used Like a Scaffold for the Generation of a Human Bioartificial Pancreas

Background: Replacing damaged organs with biological substitutes capable of protecting the islets and facilitating vascularization is a great objective in the field of islet transplantation. A decellularized placenta includes a large number of cotyledons with a conserved vessel structure of the native organ. Our goal is to obtain a perfect decellularization protocol to generate pre-vascularized organoids by recellularizing this ECM with HUVECs and pancreatic islets. Methods: After blood removal, cotyledons were dissected from the placentas and decellularized using a bioreactor. Cell removal was assessed by histology and quantification of residual DNA. Presence of structural proteins and ECM structure were analyzed using SEM, CT scan and mass spectrometry. Recellularization protocols were conducted, with HUVECs or BOECs as endothelial cell sources, and with Ins-1E cells or rat islets as insulin secreting cell sources. Function of recellularized cotyledons was assessed in vitro with glucose stimulated insulin secretion tests (GSIS). To assess in vivo biocompatibility and function of the scaffolds, we transplanted in diabetic NSG mice. Glycaemia was measured every day to monitor normalization of blood glucose levels. Results: Our protocol led to successful decellularization, as evidenced by the absence of cells and the preserved ECM structure. Moreover, DNA quantification did not reveal any residual DNA. Quantification of GAG and hydroxyproline, and mass spectrometry analysis show that structural proteins are conserved. SEM and CT scan images revealed that the ECM structure was preserved after the decellularization protocol. Cells after recellularization showed a good vascularization after 7 days. The GSIS test shows a perfect organ response in the production of insulin. Conclusions: The decellularized cotyledon is the perfect scaffold to reproduce a prevascularized insulin-producing organ, which allows transplanted cells to survive during the peri-transplantation period.

Mechanisms of Immunomodulation and Cytoprotection Conferred to Pancreatic Islet by Human Amniotic Epithelial Cells

Inhibiting pro-inflammatory cytokine activity can reverse inflammation mediated dysfunction of islet grafts. Human amniotic epithelial cells (hAECs) possess regenerative, immunomodulatory and anti-inflammatory properties. We hypothesized that hAECs could protect islets from cellular damage induced by pro-inflammatory cytokines. To verify our hypothesis, hAEC monocultures, rat islets (RI), or RI-hAEC co-cultures where exposed to a pro-inflammatory cytokine cocktail (Interferon γ: IFN-γ, Tumor necrosis factor α: TNF-α and Interleukin-1β: IL-1β). The secretion of anti-inflammatory cytokines and gene expression changes in hAECs and viability and function of RI were evaluated. The expression of non-classical Major Histocompatibility Complex (MHC) class I molecules by hAECs cultured with various IFN-γ concentrations were assessed. Exposure to the pro-inflammatory cocktail significantly increased the secretion of the anti-inflammatory cytokines IL6, IL10 and G-CSF by hAECs, which was confirmed by upregulation of IL6, and IL10 gene expression. HLA-G, HLA-E and PDL-1 gene expression was also increased. This correlated with an upregulation of STAT1, STAT3 and NF-κB1gene expression levels. RI co-cultured with hAECs maintained normal function after cytokine exposure compared to RI cultured alone, and showed significantly lower apoptosis rate. Our results show that exposure to pro-inflammatory cytokines stimulates secretion of anti-inflammatory and immunomodulatory factors by hAECs through the JAK1/2 – STAT1/3 and the NF-κB1 pathways, which in turn protects islets against inflammation-induced damages. Integrating hAECs in islet transplants appears as a valuable strategy to achieve to inhibit inflammation mediated islet damage, prolong islet survival, improve their engraftment and achieve local immune protection allowing reducing systemic immunosuppressive regimens.Graphical This study focuses on the cytoprotective effect of isolated hAECs on islets exposed to pro-inflammatory cytokines in vitro. Exposure to pro-inflammatory cytokines stimulated secretion of anti-inflammatory and immunomodulatory factors by hAECs putatively through the JAK1/2 – STAT1/3 and the NF-κB1 pathways. This had protective effect on islets against inflammation-induced damages. Taken together our results indicate that incorporating hAECs in islet transplants could be a valuable strategy to inhibit inflammation mediated islet damage, prolong islet survival, improve their engraftment and achieve local immune protection allowing to reduce systemic immunosuppressive regimens.

A bioinspired scaffold for rapid oxygenation of cell encapsulation systems

Inadequate oxygenation is a major challenge in cell encapsulation, a therapy which holds potential to treat many diseases including type I diabetes. In such systems, cellular oxygen (O2) delivery is limited to slow passive diffusion from transplantation sites through the poorly O2-soluble encapsulating matrix, usually a hydrogel. This constrains the maximum permitted distance between the encapsulated cells and host site to within a few hundred micrometers to ensure cellular function. Inspired by the natural gas-phase tracheal O2 delivery system of insects, we present herein the design of a biomimetic scaffold featuring internal continuous air channels endowed with 10,000-fold higher O2 diffusivity than hydrogels. We incorporate the scaffold into a bulk hydrogel containing cells, which facilitates rapid O2 transport through the whole system to cells several millimeters away from the device-host boundary. A computational model, validated by in vitro analysis, predicts that cells and islets maintain high viability even in a thick (6.6 mm) device. Finally, the therapeutic potential of the device is demonstrated through the correction of diabetes in immunocompetent mice using rat islets for over 6 months.

Low-grade elevation of palmitate and lipopolysaccharide synergistically induced β-cell damage via inhibition of neutral ceramidase

High concentrations of free fatty acids (FFAs) or lipopolysaccharide (LPS) could lead to β-cell apoptosis and dysfunction, while low-grade elevation of FFAs or LPS, which are more common in people with type 2 diabetes mellitus (T2DM) or obesity, have no obvious toxic effect on β-cells. Palmitate is a component closely related to metabolic disorders in FFAs. Recent studies have found that low-grade elevation of palmitate and LPS synergistically affects the sphingolipid signaling pathway by activating Toll-like receptor 4 (TLR4) and further enhances the expression of inflammatory cytokines in immune cells. Previous studies demonstrated that sphingolipids also played an important role in the occurrence and development of T2DM. This study aimed to investigate the synergistic effects of low-grade elevation of palmitate and LPS on viability, apoptosis and insulin secretion in the rat pancreatic β-cell line INS-1 or islets and the role of sphingolipids in this process. We showed that low-grade elevation of palmitate or LPS alone did not affect the viability, apoptosis, glucose-stimulated insulin secretion (GSIS) or intracellular insulin content of INS-1 cells or islets, while the combination of the two synergistically inhibited cell viability, induced apoptosis and decreased basal insulin secretion in INS-1 cells or islets. Treatment with palmitate and LPS markedly upregulated TLR4 protein expression and downregulated neutral ceramidase (NCDase) activity and protein expression. Additionally, low-grade elevation of palmitate and LPS synergistically induced a significant increase in ceramide and a decrease in sphingosine-1-phosphate. Blocking TLR4 signaling or overexpressing NCDase remarkably attenuated INS-1 cell injury induced by the combination of palmitate and LPS. However, inhibition of ceramide synthase did not ameliorate injury induced by palmitate and LPS. Overall, we show for the first time that low-grade elevation of palmitate and LPS synergistically induced β-cell damage by activating TLR4 signaling, inhibiting NCDase activity, and further modulating sphingolipid metabolism, which was different from a high concentration of palmitate-induced β-cell injury by promoting ceramide synthesis.

A combination of melatonin and moderate-intensity aerobic exercise improves pancreatic beta-cell function and glycemic homeostasis in type 2 diabetic model of animals

Nocturnal melatonin secretion is important for preservation of ß-cell mass and function. Knowing that type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder characterized by hyperglycemia caused by the elevated resistance of peripheral tissues to insulin, reduction in pineal melatonin and disturbances of insulin secretion by pancreatic ß-cells. In this context, exercise is considered one of the most valuable non-pharmacological approaches for treatment of T2DM. Considering the beneficial role of melatonin on glycemic metabolism in physical exercise, we investigated the effects of moderate-intensity aerobic exercise plus melatonin on glycemic homeostasis, the morphology and architecture of pancreas in spontaneous T2DM animals [Goto-Kakizaki (GK) rats]. The results confirmed that melatonin alone reduced the mass of epididymal white adipose tissue (WAT); however, only the combination of melatonin and physical exercise significantly reduced caloric intake, body weight, WAT and improved glucose tolerance and insulin sensitivity in T2DM rats. This combination also reduced apoptosis of cells in pancreatic islets. We observed either melatonin or the combination was able to reduce insulinemia. However, only the combination improved the morphology of the pancreatic islets. Thus, we conclude that in GK rats, melatonin plays a crucial role in the functionality of the pancreas to improve insulin sensitivity of peripheral tissues and, consequently, to maintain the glucose homeostasis. In addition, the combination is more efficiency to improve glucose tolerance and integrity of pancreatic islets in GK rats than melatonin alone.

Decreased proliferation of aged rat beta cells corresponds with enhanced expression of the cell cycle inhibitor p27KIP1.

Over 400 million people are diabetic. Type 1 and type 2 diabetes are characterized by decreased functional β-cell mass and, consequently, decreased glucose-stimulated insulin secretion. A potential intervention is transplantation of β-cell containing islets from cadaveric donors. A major impediment to greater application of this treatment is the scarcity of transplant-ready β-cells. Therefore, inducing β-cell proliferation ex vivo could be used to expand functional β-cell mass prior to transplantation. Various molecular pathways are sufficient to induce proliferation of young β-cells; however, aged β-cells are refractory to these proliferative signals. Given that the majority of cadaveric donors fit an aged demographic, defining the mechanisms that impede aged β-cell proliferation is imperative. We demonstrate that aged rat (5-month-old) β-cells are refractory to mitogenic stimuli that otherwise induce young rat (5-week-old) β-cell proliferation. We hypothesized that this change in proliferative capacity could be due to differences in cyclin-dependent kinase inhibitor expression. We measured levels of p16INK4a , p15INK4b , p18INK4c , p19INK4d , p21CIP1 , p27KIP1 and p57KIP2 by immunofluorescence analysis. Our data demonstrates an age-dependent increase of p27KIP1 in rat β-cells by immunofluorescence and was validated by increased p27KIP1 protein levels by western blot analysis. Interestingly, HDAC1, which modulates the p27KIP1 promoter acetylation state, is downregulated in aged rat islets. These data demonstrate increased p27KIP1 protein levels at 5 months of age, which may be due to decreased HDAC1 mediated repression of p27KIP1 expression. As the majority of transplant-ready β-cells come from aged donors, it is imperative that we understand why aged β-cells are refractory to mitogenic stimuli. Our findings demonstrate that increased p27KIP1 expression occurs early in β-cell aging, which corresponds with impaired β-cell proliferation. Furthermore, the correlation between HDAC1 and p27 levels suggests that pathways that activate HDAC1 in aged β-cells could be leveraged to decrease p27KIP1 levels and enhance β-cell proliferation.

Telmisartan Potentiates Insulin Secretion via Ion Channels, Independent of the AT1 Receptor and PPARγ.

Angiotensin II type 1 (AT1) receptor blockers (ARBs), as antihypertensive drugs, have drawn attention for their benefits to individuals with diabetes and prediabetes. However, the direct effects of ARBs on insulin secretion remain unclear. In this study, we aimed to investigate the insulinotropic effect of ARBs and the underlying electrophysiological mechanism. We found that only telmisartan among the three ARBs (telmisartan, valsartan, and irbesartan) exhibited an insulin secretagogue role in rat islets. Independent of AT1 receptor and peroxisome proliferator-activated receptor γ (PPARγ), telmisartan exerted effects on ion channels including voltage-dependent potassium (Kv) channels and L-type voltage-gated calcium channels (VGCCs) to promote extracellular Ca2+ influx, thereby potentiating insulin secretion in a glucose-dependent manner. Furthermore, we identified that telmisartan directly inhibited Kv2.1 channel on a Chinese hamster ovary cell line with Kv2.1 channel overexpression. Acute exposure of db/db mice to a telmisartan dose equivalent to therapeutic doses in humans resulted in lower blood glucose and increased plasma insulin concentration in OGTT. We further observed the telmisartan-induced insulinotropic and electrophysiological effects on pathological pancreatic islets or β-cells isolated from db/db mice. Collectively, our results establish an important insulinotropic function of telmisartan distinct from other ARBs in the treatment of diabetes.

Effect of kolaviron on islet dynamics in diabetic rats.

Kolaviron, a biflavonoid isolated from the edible seeds of Garcinia kola, lowers blood glucose in experimental models of diabetes; however, the underlying mechanisms are not yet fully elucidated. The objective of the current study was to assess the effects of kolaviron on islet dynamics in streptozotocin-induced diabetic rats. Using double immunolabeling of glucagon and insulin, we identified insulin-producing β- and glucagon-producing α-cells in the islets of diabetic and control rats and determined the fractional β-cell area, α-cell area and islet number. STZ challenged rats presented with islet hypoplasia and reduced β-cell area concomitant with an increase in α-cell area. Kolaviron restored some islet architecture in diabetic rats through the increased β-cell area. Overall, kolaviron-treated diabetic rats presented a significant (p < 0.05) increase in the number of large and very large islets compared to diabetic control but no difference in islet number and α-cell area. The β-cell replenishment potential of kolaviron and its overall positive effects on glycemic control suggest that it may be a viable target for diabetes treatment.

Insulin expression and insulitis degree of diabetic rats after giving sikkam leaves (Bischofia javanica Blume)

Context: Gallic acid and quercetin in sikkam leaves (Bischofia javanica) have scientific potential as antidiabetic agents. Quercetin suppresses hyperglycemia by inhibiting active glucose transport. Meanwhile, gallic acid acts as an antidiabetic is closely related to its antioxidant properties. Antioxidant compounds neutralize cells that experience oxidative stress by donating their hydrogen atoms. Aims: To analyze the degree of insulitis and insulin expression in the pancreatic cells of rats induced diabetes mellitus (DM) after giving B. javanica leaves extract. Methods: The treatment groups consisted of G0: negative control (-); G1: positive control (alloxan induction + standard feed); G2: alloxan induction + 300 mg/kg BW of B. javanica leaves ethanol extract; G3: alloxan induction + 600 mg/kg BW of B. javanica leaves ethanol extract; G4: alloxan induction + ethanol extract of B. javanica leaves 900 mg/kg BW and G5: alloxan induction + glibenclamide 4.5 mg/kg BW. At day 28, the rats were sacrificed, and the pancreatic tissue dissected. This was analyzed for degree of insulitis and insulin expression by anti-insulin antibodies using immunohistochemistry and hematoxylin-eosin. Results: There was a significant difference (p = 0.000) in insulin expression and insulitis degree. By the increase of B. javanica leaves dose, the insulin expression value also increased, and the degree of insulitis in Langerhans’ islets of DM rats was decreased. Islets of Langerhans in insulin production returned to normal after being given B. javanica ethanol extract 900 mg/kg BW like glibenclamide. Conclusions: Bischofia javanica ethanol extract increased insulin production and reduced the degree of insulitis in the islets of Langerhans histology. Further research is needed to determine the extent to which this extract protects from the sequelae of diabetes.

The Transcriptome and Epigenome Reveal Novel Changes in Transcription Regulation During Pancreatic Rat Islet Maturation.

Islet function is critical for normal glucose homeostasis. Unlike adult β cells, fetal and neonatal islets are more proliferative and have decreased insulin secretion in response to stimuli. However, the underlying mechanisms governing functional maturity of islets have not been completely elucidated. Pancreatic islets comprise different cell types. The microenvironment of islets and interactions between these cell types are critical for β-cell development and maturation. Thus, the study of intact islets is optimal to identify novel molecular mechanisms controlling islet functional development. Transcriptomes and genome-wide histone landscapes of H3K4me3, H3K27me3, and H3K27Ac from intact islets isolated from 2- and 10-week-old Sprague-Dawley rats were integrated to elucidate genes and pathways modulating islet development, as well as the contribution of epigenetic regulation. A total of 4489 differentially expressed genes were identified; 2289 and 2200 of them were up- and down-regulated in 10-week islets, respectively. Ingenuity Pathway Analysis revealed critical pathways regulating functional maturation of islets, including nutrient sensing, neuronal function, immune function, cell replication, and extracellular matrix. Furthermore, we identified significant changes in enrichment of H3K4me3, H3K27me3, and H3K27Ac marks, which correlated with expression changes of genes critical for islet function. These histone marks were enriched at critical transcription factor-binding motifs, such as Hoxa9, C/EBP-β, Gata1, Foxo1, E2f1, E2f3, and Mafb. In addition, our chromatin immunoprecipitation sequencing data revealed multiple potential bivalent genes whose poised states changed with maturation. Collectively, our current study identified critical novel pathways for mature islet function and suggested a role for histone modifications in regulating islet development and maturation.

MiR-320a induces pancreatic β cells dysfunction in diabetes by inhibiting MafF

A variety of studies indicate that microRNAs (miRNAs) are involved in diabetes. However, the direct role of miR-320a in the pathophysiology of pancreatic β cells under diabetes mellitus remains unclear. In the current study, islet transplantation and hyperglycemic clamp assays were performed in miR-320a transgenic mice to explore the effects of miR-320a on pancreatic β cells in vivo. Meanwhile, β cell-specific overexpression or inhibition of miR-320a was delivered by adeno-associated virus (AAV8). In vitro, overexpression or downregulation of miR-320a was introduced in cultured rat islet tumor cells (INS1). RNA immunoprecipitation sequencing (RIP-Seq), luciferase reporter assay, and western blotting were performed to identify the target genes. Results showed that miR-320a was increased in the pancreatic β cells from high-fat-diet (HFD)-treated mice. Overexpression of miR-320a could not only deteriorate the HFD-induced pancreatic islet dysfunction, but also initiate pancreatic islet dysfunction spontaneously in vivo. Meanwhile, miR-320a increased the ROS level, inhibited proliferation, and induced apoptosis of cultured β cells in vitro. Finally, we identified that MafF was the target of miR-320a that responsible for the dysfunction of pancreatic β cells. Our data suggested that miR-320a could damage the pancreatic β cells directly and might be a potential therapeutic target of diabetes.