Decreased Beta-cell Mass Research Articles

SRSF2 is essential for maintaining pancreatic beta-cell identity and regulating glucose homeostasis in mice

Diabetes is characterized by decreased beta-cell mass and islet dysfunction. The splicing factor SRSF2 plays a crucial role in cell survival, yet its impact on pancreatic beta cell survival and glucose homeostasis remains unclear. We observed that the deletion of Srsf2 specifically in beta cells led to time-dependent deterioration in glucose tolerance, impaired insulin secretion, decreased islet mass, an increased number of alpha cells, and the onset of diabetes by the age of 10 months in mice. Single-cell RNA sequencing (scRNA-seq) analyses revealed that, despite an increase in populations of unfolded protein response (UPR)-activated and undifferentiated beta cells within the SRSF2_KO group, there was a notable decrease in the expression of UPR-related and endoplasmic reticulum (ER)-related genes, accompanied by a loss of beta-cell identity. This suggests that beta cells have transitioned from an adaptive phase to a maladaptive phase in islets of 10-month-old SRSF2_KO mice. Further results demonstrated that deletion of SRSF2 caused decreased proliferation in beta cells within 3-month-old islets and Min6 cells. These findings underscore the essential role of SRSF2 in controlling beta-cell proliferation and preserving beta-cell function in mice.

The adhesion GPCR GPR116/ADGRF5 has a dual function in pancreatic islets regulating somatostatin release and islet development

Glucose homeostasis is maintained by hormones secreted from different cell types of the pancreatic islets and controlled by manifold input including signals mediated through G protein-coupled receptors (GPCRs). RNA-seq analyses revealed expression of numerous GPCRs in mouse and human pancreatic islets, among them Gpr116/Adgrf5. GPR116 is an adhesion GPCR mainly found in lung and required for surfactant secretion. Here, we demonstrate that GPR116 is involved in the somatostatin release from pancreatic delta cells using a whole-body as well as a cell-specific knock-out mouse model. Interestingly, the whole-body GPR116 deficiency causes further changes such as decreased beta-cell mass, lower number of small islets, and reduced pancreatic insulin content. Glucose homeostasis in global GPR116-deficient mice is maintained by counter-acting mechanisms modulating insulin degradation. Our data highlight an important function of GPR116 in controlling glucose homeostasis.

Footprint of pancreas infiltrating and circulating immune cells throughout type 1 diabetes development.

Type 1 diabetes (T1D) is defined by immune cell infiltration of the pancreas, in particular the islets of Langerhans, referred to as insulitis, which is especially prominent during the early disease stages in association with decreased beta cell mass. An in-depth understanding of the dynamics and phenotype of the immune cells infiltrating the pancreas and the accompanying changes in their profiles in peripheral blood during T1D development is critical to generate novel preventive and therapeutic approaches, as well as to find biomarkers for the disease process. Using multi-parameter flow cytometry, we explored the dynamic changes of immune cells infiltrating the pancreas and the pancreatic draining lymph nodes (PLN), compared to those in peripheral blood in female and male non-obese diabetic (NOD) mice during T1D progression. The early stages of T1D development were characterized by an influx of innate dendritic cells and neutrophils in the pancreas. While dendritic cells seemed to move in and out (to the PLN), neutrophils accumulated during the pre-symptomatic phase and reached a maximum at 8 weeks of age, after which their numbers declined. During disease progression, CD4+ and CD8+ T cells appeared to continuously migrate from the PLN to the pancreas, which coincided with an increase in beta cell autoimmunity and insulitis severity, and a decline in insulin content. At 12 weeks of age, CD4+ and especially CD8+ T cells in the pancreas showed a dramatic shift from naïve to effector memory phenotype, in contrast to the PLN, where most of these cells remained naïve. A large proportion of pancreas infiltrating CD4+ T cells were naïve, indicating that antigenic stimulation was not necessary to traffic and invade the pancreas. Interestingly, a pre-effector-like T cell dominated the peripheral blood. These cells were intermediates between naïve and effector memory cells as identified by single cell RNA sequencing and might be a potential novel therapeutic target. These time- and tissue-dependent changes in the dynamics and functional states of CD4+ and CD8+ T cells are essential steps in our understanding of the disease process in NOD mice and need to be considered for the interpretation and design of disease-modifying therapies.

OR12-02 NRF2 Is Essential For Expanding Pancreatic Beta-Cell Mass During Pregnancy

Abstract Disclosure: S. Baumel-Alterzon: None. A. Garcia-Ocana: None. D.K. Scott: None. The late stages of the mammalian pregnancy are accompanied with increased insulin resistance due to the increased glucose demand of the growing fetus. Therefore, as a compensatory response, in order to maintain the maternal normal blood glucose levels, maternal beta-cell mass expands leading to increased insulin release. Beta-cell proliferation, beta-cell neogenesis, and decreased beta-cell apoptosis, are major contributors for the beta-cell adaptive response during pregnancy. Defects in these cellular processes can lead to gestational diabetes mellitus (GDM). GDM, which occurs in ∼7-8% of the total pregnancy cases within the U.S, results in adverse outcomes for both the mother and newborn. This highlights the urgent need for exploring the yet undefined intracellular mechanisms that regulate functional beta-cell mass expansion during pregnancy. GWAS analysis revealed several mutations in the Nrf2 antioxidant pathway that are associated with diabetes. Additionally, we previously found that Nrf2 is required for adaptive beta-cell expansion after high fat feeding via stimulation of beta-cell proliferation, increased beta-cell survival, and maintenance of beta-cell identity. Interestingly, Nrf2 expression levels are increased by 4.3-fold (+/- 6.60, p<0.05, n=3-4) in maternal beta-cells during pregnancy, peaking at gestational day (GD)19, while returning to normal levels postpartum. Based on these observations, we hypothesize that Nrf2 might play an important role in expanding beta-cell mass during pregnancy and that disruption of Nrf2 expression or function might lead to GDM. In order to test our hypothesis, we generated tamoxifen (Tam)-induced beta cell-specific deletion of Nrf2 in female mice by crossing MIP-CreERTTAM with Nrf2lox/lox mice (βNrf2KO). In order to exclude any effect of Tam on pregnancy outcomes, females mice were injected with Tam (or vehicle control) a month before gestation. Pancreata were harvested at GD15 or GD19 and immunolabeled with Ki67 and insulin (proliferation assay), TUNEL and insulin (apoptotic assay) or insulin alone (beta-cell mass analysis). Our results show that βNrf2KO mice exhibit 70% reduction (+/- 0.25, p<0.01, n=4-6) in beta-cell proliferation and a 0.13% increase in beta-cell death (+/- 0.03, p<0.05, n=4-6) at GD15 compared to littermate controls. Importantly, βNrf2KO pregnant mice display a remarkable decrease in beta-cell mass at GD19 (50%) (+/- 0.68, p<0.05, n=4) compared with control mice, accompanied by a trending decrease in plasma insulin and increase in blood glucose. No changes were observed in the average number of embryos between βNrf2KO and control pregnant mice. We conclude that Nrf2 is required for beta-cell mass expansion during pregnancy by controlling beta-cell proliferation and survival and alterations in Nrf2 action could potentially lead to development of GDM. Presentation: Friday, June 16, 2023

Mechanisms of Beta-Cell Apoptosis in Type 2 Diabetes-Prone Situations and Potential Protection by GLP-1-Based Therapies.

Type 2 diabetes (T2D) is characterized by chronic hyperglycemia secondary to the decline of functional beta-cells and is usually accompanied by a reduced sensitivity to insulin. Whereas altered beta-cell function plays a key role in T2D onset, a decreased beta-cell mass was also reported to contribute to the pathophysiology of this metabolic disease. The decreased beta-cell mass in T2D is, at least in part, attributed to beta-cell apoptosis that is triggered by diabetogenic situations such as amyloid deposits, lipotoxicity and glucotoxicity. In this review, we discussed the molecular mechanisms involved in pancreatic beta-cell apoptosis under such diabetes-prone situations. Finally, we considered the molecular signaling pathways recruited by glucagon-like peptide-1-based therapies to potentially protect beta-cells from death under diabetogenic situations.

The Effect of Cholesterol on Membrane-Bound Islet Amyloid Polypeptide.

Islet amyloid polypeptide (IAPP) is a proposed cause of the decreased beta-cell mass in patients with type-II diabetes. The molecular composition of the cell-membrane is important for regulating IAPP cytotoxicity and aggregation. Cholesterol is present at high concentrations in the pancreatic beta-cells, and in-vitro experiments have indicated that it affects the amyloid formation of IAPP either by direct interactions or by changing the properties of the membrane. In this study we apply atomistic, unbiased molecular dynamics simulations at a microsecond timescale to investigate the effect of cholesterol on membrane bound IAPP. Simulations were performed with various combinations of cholesterol, phosphatidylcholine (PC) and phosphatidylserine (PS) lipids. In all simulations, the helical structure of monomer IAPP was stabilized by the membrane. We found that cholesterol decreased the insertion depth of IAPP compared to pure phospholipid membranes, while PS lipids counteract the effect of cholesterol. The aggregation propensity has previously been proposed to correlate with the insertion depth of IAPP, which we found to decrease with the increased ordering of the lipids induced by cholesterol. Cholesterol is depleted in the vicinity of IAPP, and thus our results suggest that the effect of cholesterol is indirect.

2124-P: Raptor Levels Are Critical in the Adaptation of Beta Cells to High-Fat Diet

mTORC1 signaling is a central regulator of autophagy and altered autophagic activity has been implicated in the beta-cells of patients with type 2 diabetes, and in the beta-cells of obese mice. The importance of mTORC1 signaling and Raptor in beta-cells has been recently demonstrated using raptor knock-out models (βraKO mice). These studies have shown that complete deletion of Raptor impairs beta-cell proliferation and survival of the beta-cell, and both in function and insulin processing. However, nothing is known whether reduced mTORC1 levels impair beta-cell adaptation to diabetogenic conditions. Therefore, we decided to study mice with haploinsufficiency of Raptor in beta-cells (βraHET). Mice with heterozygous deletion of Raptor in beta-cells were generated by crossing raptorf/f with Rip-Cre mice (βraHET). βraHET and control mice were fed regular chow (RC) or high-fat diet (HFD). Mice fed with RC showed no difference at the metabolic level, islets morphology, or beta-cell function. βraHET mice fed HFD for eight weeks showed an impaired Intraperitoneal Glucose Tolerance Test, fed insulin secretion and a decreased beta-cell mass, compared to control mice. Isolated islets from βraHET mice fed HFD for eight weeks showed impaired insulin secretion either measured by perifusion or static incubation compared to control islets. Next, we tested whether isolated islets from βraHET mice are more susceptible to beta-cell stressors. Beta-cell death determined by TUNEL and cleavage Caspase 3 was increased (50 and 60%) in the βraHET islets after 24 h exposure to palmitate. In addition, autophagy markers such as LC3 and p62 were increased in islets from βraHET treated with palmitate. The increase in cell death and autophagy is specific to lipotoxicity since there βraHET islets exhibited no increase in cleavage Caspase 3 in TUNEL when they were treated with proinflammatory cytokines or ER Stress inducers. In conclusion, we demonstrate that adequate levels of Raptor are critical in the adaptation of the beta-cell to HFD. Disclosure M. Blandino-Rosano: None. E. Bernal-Mizrachi: None.

Decreased pancreatic acinar cell number in type 1 diabetes.

Individuals with longstanding and recent-onset type 1 diabetes have a smaller pancreas. Since beta cells represent a very small portion of the pancreas, the loss of pancreas volume in diabetes is primarily due to the loss of pancreatic exocrine mass. However, the structural changes in the exocrine pancreas in diabetes are not well understood. To characterise the pancreatic endocrine and exocrine compartments in diabetes, we studied pancreases from adult donors with type 1 diabetes compared with similarly aged donors without diabetes. Islet cell mass, islet morphometry, exocrine mass, acinar cell size and number and pancreas fibrosis were assessed by immunohistochemical staining. To better understand possible mechanisms of altered pancreas size, we measured pancreas size in three mouse models of insulin deficiency. Pancreases from donors with type 1 diabetes were approximately 45% smaller than those from donors without diabetes (47.4 ± 2.6 vs 85.7 ± 3.7g), independent of diabetes duration or age of onset. Diabetic donor pancreases had decreased beta cell mass (0.061 ± 0.025 vs 0.94 ± 0.21g) and reduced total exocrine mass (42.0 ± 4.9 vs 96.1 ± 6.5g). Diabetic acinar cells were similar in size but fewer in number compared with those in pancreases from non-diabetic donors (63.7 ± 8.1 × 109 vs 121.6 ± 12.2 × 109 cells/pancreas), likely accounting for the difference in pancreas size. Within the type 1 diabetes exocrine tissue, there was a greater degree of fibrosis. The pancreases in three mouse models of insulin deficiency were similar in size to those in control mice. Pancreases from donors with type 1 diabetes are smaller than normal donor pancreases because exocrine cells are fewer in number rather than smaller in size; these changes occur early in the disease process. Our mouse data suggest that decreased pancreas size in type 1 diabetes is not directly caused by insulin deficiency, but the precise mechanism responsible remains unclear.

Beta-cell β1 integrin deficiency affects in utero development of islet growth and vascularization.

The β1 integrin subunit contributes to pancreatic beta cell growth and function through communication with the extracellular matrix (ECM). The effects of in vitro and in vivo β1 integrin knockout have been extensively studied in mature islets, yet no study to date has examined how the loss of β1 integrin during specific stages of pancreatic development impacts beta cell maturation. Beta-cell-specific tamoxifen-inducible Cre recombinase (MIP-CreERT) mice were crossed with mice containing floxed Itgb1 (β1 integrin) to create an inducible mouse model (MIPβ1KO) at the second transition stage (e13.5)of pancreas development. By e19.5-20.5, the expression of beta-cell β1 integrin in fetal MIPβ1KO mice was significantly reduced and these mice displayed decreased beta cell mass, density and proliferation. Morphologically, fetal MIPβ1KO pancreata exhibited reduced islet vascularization and nascent endocrine cells in the ductal region. In addition, decreased ERK phosphorylation was observed in fetal MIPβ1KO pancreata. The expression of transcription factors needed for beta-cell development was unchanged in fetal MIPβ1KO pancreata. The findings from this study demonstrate that β1 integrin signaling is required during a transition-specific window in the developing beta-cell to maintain islet mass and vascularization.

2190-P: Sex-Based Differences in Nr4a1 Beta-Cell Activity

The orphan nuclear hormone receptor Nr4a1 plays a critical role in maintaining and expanding functional beta cell mass. Overexpression of Nr4a1 is sufficient to induce beta cell proliferation in primary beta cells by upregulating cell cycle progression, while maintaining glucose-stimulated insulin secretion. Full body deletion of Nr4a1 results in decreased beta cell mass, while resulting in modified fuel utilization in liver and muscle. Knock down of Nr4a1 decreases glycolytic and TCA cycle gene expression, impairs mitochondrial respiration, decreases cellular ATP levels, and ultimately decreases glucose-stimulated insulin secretion. While Nr4a1 is currently considered an orphaned nuclear hormone receptor, recent findings suggest that unsaturated fatty acids may function as a ligand, and that exposure to these fatty acid’s changes Nr4a1 affinity for promoters of genes involved in fuel utilization. To determine the effect of Nr4a1 presence of absence in the beta cell, and the effect of fatty acid exposure, we developed a beta cell specific, inducible, Nr4a1 knock out mouse. Male and female mice, deficient for Nr4a1 beginning at 3 months of age, were fed a normal chow or high fat diet for an additional four months. Here we present data demonstrating a sex specific difference in phenotype. Male mice demonstrated improved glucose tolerance at four months of feeding, while female mice demonstrated impaired glucose tolerance as early as one month of feeding. Here we present data regarding differences in beta cell mass, glucose stimulated insulin secretion, as well as Nr4a1 DNA binding from in islets from male and female mice exposed to unsaturated and saturated fatty acids. These data begin to demonstrate the role of Nr4a1 in controlling gene expression as a function of nutrient exposure, as well as sex-based differences in Nr4a1 activity. Disclosure J.S. Tessem: None. J. Herring: None. C.J. Smith: None. M.C. Austin: None. A.G. Wynn: None. Funding American Diabetes Association (1-17-IBS-101 to J.S.T.)

Increased mast cell number is associated with a decrease in beta-cell mass and regeneration in type 2 diabetic rats

The role of mast cells (MCs) in type 2 diabetes (T2D) is not thoroughly studied as much as in T1D. Therefore in the current study we investigated correlation between these cells and various parameters of islets of Langerhans (IOL) in rats which were equally divided (n = 40) into; control and diabetic groups.We detected a significantly increased (p < 0.05) MC count (MCC) in the diabetic IOL compared to the control, together with a noticeable intra-islet seeding of these cells which displayed a tryptase positive immunostaining.A significant positive correlation (p < 0.05) between MCC and the % of glucagon cells per islet was detected in DM, unlike mass of the islets, mass of β-cells, and % of β-cells per islet which were negatively correlated with MCC. Similarly, there was a negative correlation between MCC with β-cell proliferation and neogenesis frequency in DM. This highlights the potential association between the increased MC number and the diminished islet`s mass as well as regeneration which may fasten the progression of T2D.

Integrative Analysis of Genome and Expression Profile Data Reveals the Genetic Mechanism of the Diabetic Pathogenesis in Goto Kakizaki (GK) Rats.

The Goto Kakizaki (GK) rats which can spontaneously develop type 2 diabetes (T2D), are generated by repeated inbreeding of Wistar rats with glucose intolerance. The glucose intolerance in GK rat is mainly attributed to the impairment in glucose-stimulated insulin secretion (GSIS). In addition, GK rat display a decrease in beta cell mass, and a change in insulin action. However, the genetic mechanism of these features remain unclear. In the present study, we analyzed the population variants of GK rats and control Wistar rats by whole genome sequencing and identified 1,839 and 1,333 specific amino acid changed (SAAC) genes in GK and Wistar rats, respectively. We also detected the putative artificial selective sweeps (PASS) regions in GK rat which were enriched with GK fixed variants and were under selected in the initial diabetic-driven derivation by homogeneity test with the fixed and polymorphic sites between GK and Wistar populations. Finally, we integrated the SAAC genes, PASS region genes and differentially expressed genes in GK pancreatic beta cells to reveal the genetic mechanism of the impairment in GSIS, a decrease in beta cell mass, and a change in insulin action in GK rat. The results showed that Slc2a2 gene was related to impaired glucose transport and Adcy3, Cacna1f, Bmp4, Fam3b, and Ptprn2 genes were related to Ca2+ channel dysfunction which may responsible for the impaired GSIS. The genes Hnf4g, Bmp4, and Bad were associated with beta cell development and may be responsible for a decrease in beta cell mass while genes Ide, Ppp1r3c, Hdac9, Ghsr, and Gckr may be responsible for the change in insulin action in GK rats. The overexpression or inhibition of Bmp4, Fam3b, Ptprn2, Ide, Hnf4g, and Bad has been reported to change the glucose tolerance in rodents. However, the genes Bmp4, Fam3b, and Ptprn2 were found to be associated with diabetes in GK rats for the first time in the present study. Our findings provide a comprehensive genetic map of the abnormalities in GK genome which will be helpful in understand the underlying genetic mechanism of pathogenesis of diabetes in GK rats.

Pemberian ekstrak etanol Cordyceps militaris dapat menurunkan kadar glukosa darah dan menghambat penurunan massa sel beta pankreas pada tikus diabetes mellitus yang diinduksi dengan Streptozotocin dan Nikotinamid

Background: The antioxidant and anti-inflammatory effects of Cordyceps militaris extracts can neutralize the increased production of Reactive Oxygen Species (ROS) due to hyperglycemia conditions, as well as neutralize NO produced through inducible Nitric Oxide Synthase (iNOS). Thus, the antioxidant effects of C. militaris extract are expected to decrease DNA damage and ER stress leading to intrinsic apoptosis of pancreatic beta cells. Another positive effect of C. militaris which is expected to suppress glucotoxicity is its hypoglycemic effect.Methods: The design of this study was Randomized Post-test Only Control Group Design. The study was conducted on mice divided into three groups: negative control group (DM + placebo), treatment 1 group and treatment 2 group.Results: Based on study on 15 rats, each 5 rats per group, it was found that the mean blood glucose level in control group was 399,20 ± 28,07 mg/dl, mean of blood glucose level of treatment 1group (extraction 50% ethanol Cordyceps militaris) was 198.60 ± 30.42 mg/dl, and, the mean blood glucose levels of the treatment 1 group (extract ethanol Cordyceps militaris 100%) was 141.00 ± 17.03 mg/dl. With One Way ANOVA test found that p-value = 0,001. Immunohistochemical examination result shows that beta cell mass is more prominent in either treatment group 1 and treatment group 2 compare to the control group.Conclusions: There were significant differences in blood glucose levels after extracted ethanol Cordyceps militaris. It is further known that ethanol extract of Cordyceps militaris can decrease blood glucose level by 50,25% at 50% concentration and 64,68% at 100% concentration. Immunohistochemical examination result shows that ethanol extract of C. militaris seem could inhibit the decrease of beta cell mass especially at 100% concentration.

Investigation on the Protective Effect of Canagliflozin on Pancreatic Beta-Cell Mass Using SPECT/CT Imaging with 111In-Labeled Exendin-4

Background: Defects in insulin secretory function can be divided into two elements, the dysfunction of individual beta cells and a decrease of beta cell mass (BCM). Non-invasive methods to quantify BCM have not yet been established, and thus it is not possible to observe longitudinal changes in BCM. We developed [Lys12(111In-BnDTPA-Ahx)]exendin-4 ([111In]Ex4) as a radioactive probe targeting to the glucagon-like peptide-1 receptor with the aim of quantifying BCM by the measurement of probe accumulation in the pancreas. In this study, we investigated the protective effect of sodium-glucose co-transporter-2 (SGLT2) inhibitor on BCM using in-vivo SPECT/CT imaging with this probe. Aim: We tried to investigate longitudinal change in BCM of diabetic model mice administered with SGLT2 inhibitor using in-vivo SPECT/CT imaging with [111In]Ex4. Method: Male db/db mice were assigned into two groups: canagliflozin-administered group (group A) and non-administration group (group B). In-vivo SPECT/CT scans were performed before and after the intervention. The pancreatic uptake of [111In]Ex4 was evaluated using ROI with &amp;gt; 40% of the volume of the whole pancreas with the exception of the perirenal space. Result: The pancreatic uptake was significantly decreased in group B, on the other hand, no significant change was observed in group A. After the intervention, the pancreatic uptake was significantly higher in group A than group B. Discussion: SPECT/CT imaging analysis indicated that BCM was significantly larger in group A than group B after the intervention. It is considered that this difference was made by the protective effect of canagliflozin on BCM. Conclusion: It is considered that the protective effect of canagliflozin on BCM could be investigated using in-vivo SPECT/CT imaging with [111In]Ex4. Disclosure K. Hamamatsu: None. H. Fujimoto: None. N. Fujita: None. T. Murakami: None. M. Shiotani: Employee; Self; Mitsubishi Tanabe Pharma Corporation. H. Saji: Research Support; Self; Nihon Medi-Physics Co.,Ltd. N. Inagaki: Research Support; Self; Daiichi Sankyo Company, Limited, AstraZeneca, Ono Pharmaceutical Co., Ltd., Sanofi K.K., Mitsubishi Tanabe Pharma Corporation, Takeda Pharmaceutical Company Limited, Japan Tobacco Inc., MSD K.K., Sumitomo Dainippon Pharma Co., Ltd., Nippon Boehringer Ingelheim Co. Ltd., Novartis Pharma K.K., Kissei Pharmaceutical Co., Ltd., Novo Nordisk Pharma Ltd., Eli Lilly and Company, Sanwa Chemical Industrial Co., Ltd., Teijin Pharma Limited.

Autophagy protects pancreatic beta cell mass and function in the setting of a high-fat and high-glucose diet

Autophagy is a major regulator of pancreatic beta cell homeostasis. Altered autophagic activity has been implicated in the beta cells of patients with type 2 diabetes, and in the beta cells of obese diabetic rodents. Here, we show that autophagy was induced in beta cells by either a high-fat diet or a combined high-fat and high-glucose diet, but not by high-glucose alone. However, a high-glucose intake alone did increase beta cell mass and insulin secretion moderately. Depletion of Atg7, a necessary component of the autophagy pathway, in beta cells by pancreatic intra-ductal AAV8-shAtg7 infusion in C57BL/6 mice, resulted in decreased beta cell mass, impaired glucose tolerance, defective insulin secretion, and increased apoptosis when a combined high-fat and high-glucose diet was given, seemingly due to suppression of autophagy. Taken together, our findings suggest that the autophagy pathway may act as a protective mechanism in pancreatic beta cells during a high-calorie diet.

Circulating microRNA-375 as biomarker of pancreatic beta cell death and protection of beta cell mass by cytoprotective compounds

ObjectivePrevious studies demonstrated that circulating microRNA-375 (miR-375) is a suitable plasma biomarker for real-time detection of beta cell death. The present study evaluated the use of this biomarker to assess the beta cytoprotective effect of phenylpropenoic acid glucoside (PPAG), which was previously demonstrated to protect beta cells against various types of injury, and of exendin-4, which is an established antidiabetic drug.MethodsPPAG or exendin-4 were administered in mice treated with streptozotocin (STZ) to acutely induce beta cell death. Beta cell mass and apoptotic death were measured in pancreatic tissue sections. Circulating miR-375 was measured in blood plasma by RT-qPCR. The release of miR-375 was also measured in vitro by MIN-6 beta cells.ResultsAdministration of STZ resulted in measurable circulating levels of miR-375, a decrease in beta cell mass and increase in frequency of apoptotic beta cells. In vitro, there was a good correlation between miR-375 release and the extent of beta cell death. Treatment of mice with PPAG or exendin-4 significantly attenuated STZ-induced loss of beta cell mass and beta cell apoptosis, and normalized the blood level of miR-375.ConclusionsThese findings show the potential use of serological miR-375 measurements to evaluate the beta cytoprotective effect of (potential) antidiabetic drugs in vivo.

ERK1 is dispensable for mouse pancreatic beta cell function but is necessary for glucose-induced full activation of MSK1 and CREB.

Insufficient insulin secretion from pancreatic beta cells, which is associated with a decrease in beta cell mass, is a characteristic of type 2 diabetes. Extracellular signal-related kinase 1 and 2 (ERK1/2) inhibition in beta cells has been reported to affect insulin secretion, gene transcription and survival, although whether ERK1 and ERK2 play distinct roles is unknown. The aim of this study was to assess the individual roles of ERK1 and ERK2 in beta cells using ERK1 (also known as Mapk3)-knockout mice (Erk1 -/- mice) and pharmacological approaches. NAD(P)H, free cytosolic Ca2+ concentration and insulin secretion were determined in islets. ERK1 and ERK2 subplasmalemmal translocation and activity was monitored using total internal reflection fluorescence microscopy. ERK1/2, mitogen and stress-activated kinase1 (MSK1) and cAMP-responsive element-binding protein (CREB) activation were evaluated by western blot and/or immunocytochemistry. The islet mass was determined from pancreatic sections. Glucose induced rapid subplasmalemmal recruitment of ERK1 and ERK2. When both ERK1 and ERK2 were inhibited simultaneously, the rapid transient peak of the first phase of glucose-induced insulin secretion was reduced by 40% (p<0.01), although ERK1 did not appear to be involved in this process. By contrast, ERK1 was required for glucose-induced full activation of several targets involved in beta cell survival; MSK1 and CREB were less active in Erk1 -/- mouse beta cells (p<0.01) compared with Erk1 +/+ mouse beta cells, and their phosphorylation could only be restored when ERK1 was re-expressed and not when ERK2 was overexpressed. Finally, the islet mass of Erk1 -/- mice was slightly increased in young animals (4-month-old mice) vs Erk1 +/+ mice (section occupied by islets [mean±SEM]: 0.74%±0.03% vs 0.62%±0.04%; p<0.05), while older mice (10months old) were less prone to age-associated pancreatic peri-insulitis (infiltrated islets [mean±SEM]: 7.51%±1.34% vs 2.03%±0.51%; p<0.001). ERK1 and ERK2 play specific roles in beta cells. ERK2 cannot always compensate for the lack of ERK1 but the absence of a clear-cut phenotype in Erk1 -/- mice shows that ERK1 is dispensable in normal conditions.

GLP-1 signalling compensates for impaired insulin signalling in regulating beta cell proliferation in βIRKO mice.

We aimed to investigate potential interactions between insulin and glucagon-like peptide (GLP)-1 signalling pathways in the regulation of beta cell-cycle dynamics in vivo, in the context of the therapeutic potential of GLP-1 to modulate impaired beta cell function. Beta cell-specific insulin receptor knockout (βIRKO) mice, which exhibit beta cell dysfunction and an age-dependent decrease in beta cell mass, were treated with the dipeptidyl peptidase-4 inhibitor vildagliptin. Following this, glucose homeostasis and beta cell proliferation were evaluated and underlying molecular mechanisms were investigated. The sustained elevation in circulating GLP-1 levels, caused by treatment of the knockout mice with vildagliptin for 6weeks, significantly improved glucose tolerance secondary to enhanced insulin secretion and proliferation of beta cells. Treating βIRKO beta cell lines with the GLP-1 analogue, exendin-4, promoted Akt phosphorylation and protein expression of cyclins A, D1 and E two- to threefold, in addition to cyclin D2. Pancreases from the vildagliptin-treated βIRKO mice exhibited increased cyclin D1 expression, while cyclin D2 expression was impaired. Activation of GLP-1 signalling compensates for impaired growth factor (insulin) signalling and enhances expression of cyclins to promote beta cell proliferation. Together, these data indicate the potential of GLP-1-related therapies to enhance beta cell proliferation and promote beneficial outcomes in models with dysfunctional beta cells.

Npas4 Transcription Factor Expression Is Regulated by Calcium Signaling Pathways and Prevents Tacrolimus-induced Cytotoxicity in Pancreatic Beta Cells

Cytosolic calcium influx activates signaling pathways known to support pancreatic beta cell function and survival by modulating gene expression. Impaired calcium signaling leads to decreased beta cell mass and diabetes. To appreciate the causes of these cytotoxic perturbations, a more detailed understanding of the relevant signaling pathways and their respective gene targets is required. In this study, we examined the calcium-induced expression of the cytoprotective beta cell transcription factor Npas4. Pharmacological inhibition implicated the calcineurin, Akt/protein kinase B, and Ca(2+)/calmodulin-dependent protein kinase signaling pathways in the regulation of Npas4 transcription and translation. Both Npas4 mRNA and protein had high turnover rates, and, at the protein level, degradation was mediated via the ubiquitin-proteasome pathway. Finally, beta cell cytotoxicity of the calcineurin inhibitor and immunosuppressant tacrolimus (FK-506) was prevented by Npas4 overexpression. These results delineate the pathways regulating Npas4 expression and stability and demonstrate its importance in clinical settings such as islet transplantation.

Beta cell regeneration after single-round immunological destruction in a mouse model.

Aims/hypothesisAchieving a better understanding of beta cell regeneration after immunological destruction is crucial for the development of immunotherapy approaches for type 1 diabetes. In previous type 1 diabetes models, sustained immune activation eliminates regenerating beta cells, thus limiting the study of the regenerative capacity of beta cells upon immunological destruction. Here, we employed an adeno-associated virus 8 (AAV8) vector for beta cell-targeted overexpression of a foreign antigen to induce single-round immunological destruction of existing beta cells.MethodsYoung and aged C57BL/6J mice were treated with AAV8 vectors expressing the foreign antigen luciferase. Islet inflammation and regeneration was observed at 3, 6, 10 and 22 weeks post-AAV delivery.ResultsIn young C57BL/6J mice, robust humoral and cellular immune responses were developed towards antigen-expressing beta cells, leading to decreased beta cell mass. This was followed by beta cell mass replenishment, along with enhanced proliferation of insulin-positive cells, recruitment of nestin/CD34-positive endothelial cells, displacement of alpha cells and mobilisation of cytoplasmic neurogenin 3-positive cells. Mice with recovering beta cells showed normal or reduced fasting blood glucose levels and faster glucose clearance than controls. Although aged mice demonstrated similar responses to the treatment, they initially exhibited notable islet scarring and fluctuations in blood glucose levels, indicating that beta cell regeneration is slower in aged mice.Conclusions/interpretationOur hit-and-run, beta cell-targeted antigen expression system provides an opportunity to monitor the impact of single-round immunological beta cell destruction in animals with diverse genetic backgrounds or ageing status.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-014-3416-4) contains peer-reviewed but unedited supplementary material, which is available to authorised users.