Islets最新文献

HNF4α is important for beta cells' ability to adequately secrete insulin in response to glucose concentration and endoplasmic reticulum (ER) homeostasis. In humans, HNF4α mutations are responsible for Diabetes mellitus subtype MODY1, which has an age-determining onset. Additionally, in other forms of DM, there is evidence that sex can influence beta cell dysfunction, with possible involvement of ER stress pathways. Thus, we assessed the influence of sex and age on beta-cell dysfunction induced by HNF4α absence. We used an animal model with specific beta cells KO of HNF4α, induced after birth (Ins. CRE HNF4α^loxP/loxP). Glucose intolerance is observed after 10 d of KO induction, at 50 d of age, with KO males (MKO) displaying more severe glucose intolerance than KO females (FKO). The percentage of insulin-positive cells in KO mice islets is lower compared to Control at all ages evaluated, with MKO mice showing a more pronounced decline at later ages compared to FKO. Both KO groups exhibited reduced beta cell mass and increased α-cell mass, which was more pronounced in MKO. ER stress was induced in both KO groups; however, ER stress-mediated apoptosis was observed only in MKO. FKO mice show evidence of beta cell differentiated state loss. In summary, beta cell loss in HNF4α-KO is influenced by sex and age, involves induction of ER stress, and is more severe in males, where ER stress-induced beta cell death is observed. Partial protection observed in females seems to involve dedifferentiation of beta cells.

Pancreatic β-cells release insulin in response to fluctuations in plasma glucose, amino acids, and free fatty acids (FFA). Clonal cell lines and isolated islets serve as essential early models for studying the impact of nutrients and evaluating potential therapies to address β-cell dysfunction. Acute and chronic changes in FFA levels have been shown to have positive and negative effects on β-cell function both in vivo and in vitro. A key problem in comparing islet lipid studies from different laboratories is that a wide variety of methods are used to isolate, culture, and assess islet function. The current study compares bovine serum albumin (BSA) types and lipid preparation methods in clonal 832/13 cells and human islets. Changing the percentage and culture conditions when using FFA-free BSA can negatively affect β-cell function compared to regular BSA. Preparing palmitate with FFA-free BSA can rescue insulin secretion compared to treating cells alone with FFA-free BSA. Different methods of preparing palmitate can have unique effects on insulin secretion. Overall, interpreting the effects of lipids on β-cell function is complicated by a number of variables that need to be controlled for in islet experiments.

Pancreatic and duodenal homeobox protein (PDX)1 is a major transcription factor for the regulation of insulin, glucagon and somatostatin (SST) expression. PDX1 is phosphorylated by CK2 and inhibition of this kinase results in an increased insulin and decreased glucagon secretion. Therefore, we speculated in this study that CK2 also affects SST expression. To test this, we analyzed the effects of the two CK2 inhibitors CX-4945 and SGC as well as of PDX1 overexpression on SST expression and secretion in RIN14B cells by qRT-PCR, luciferase assays, Western blot and ELISA. SST expression and secretion were additionally assessed in isolated murine and human islets exposed to the CK2 inhibitors. Moreover, we determined the expression and secretion of the pancreatic endocrine hormones in CX-4945-treated mice. We found a suppressed SST expression in RIN14B cells due to a methylated SST promoter, which could be abolished by DNA demethylation. Under these conditions, we showed that CK2 inhibition increases SST gene expression and secretion. Additional experiments with overexpression of a CK2-phosphorylation mutant of PDX1 verified that SST expression is regulated by CK2. The exposure of isolated murine and human islets to CX-4945 or SGC as well as the treatment of mice with CX-4945 revealed that CK2 also regulates SST expression under physiological conditions. Taken together, these findings not only demonstrate that CK2 controls SST expression in pancreatic δ-cells but also emphasize the crucial role of this kinase in regulating the main hormones of the endocrine pancreas.

Background: We previously showed that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD - a persistent organic pollutant) activates the aryl hydrocarbon receptor (AHR) in pancreatic islets. The AHR is known to crosstalk with hypoxia-inducible factor 1α (HIF1α) in other cell types but AHR-HIF1α crosstalk has not been previously examined in islet cells. Islet cell function is sensitive to hypoxia; we hypothesize that AHR activation by environmental pollutant(s) will interfere with the HIF1α pathway response in islets, which may be detrimental to islet cell function and survival during periods of hypoxia.

Methods: We assessed AHR-HIF1α crosstalk by treating human donor islets and stem cell-derived islets (SC-islets) with 10 nM TCDD ± 1% O₂ and measuring gene expression of downstream targets of AHR (e.g. CYP1A1) and HIF1α (e.g. HMOX1).

Results: In SC-islets, co-treatment with TCDD + hypoxia consistently suppressed CYP1A1 induction compared with TCDD treatment alone. In human islets, TCDD + hypoxia co-treatment suppressed CYP1A1 induction, but only in 2 of 6 donors. Both SC-islets and human donor islets displayed hypoxia-mediated suppression of glucose-6-phosphate catalytic subunit 2 (G6PC2) expression. Glucose-stimulated insulin secretion (GSIS) in human donor islets was impaired by hypoxia exposure, but unaffected by TCDD exposure.

Conclusion: Our study shows consistent AHR-HIF1α crosstalk in SC-islets and variable crosstalk in primary human islets, depending on the donor. In both cell models, hypoxia exposure interfered with activation of the AHR pathway by TCDD but there was no evidence that AHR activation interfered with the HIF1α pathway. In summary, our data show that co-exposure to an environmental pollutant and hypoxia results in molecular crosstalk in islets.

Statins are widely used to treat hyperlipidemia and atherosclerotic cardiovascular diseases (ACVD) by significantly lowering low-density lipoprotein cholesterol (LDL-C) levels. However, their use has been associated with an increased risk of type 2 diabetes (T2D), a paradox given their lipid-lowering benefits. This study investigates the role of LDL receptors (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) in the diabetogenic effects of atorvastatin on pancreatic β-cells. Using the MIN6 pancreatic β-cell line, we assessed the impact of atorvastatin on LDL-C uptake, PCSK9 expression, glucose-stimulated insulin release (GSIR), and cell proliferation. Cellular cholesterol assays, EdU labeling, Dil-LDL uptake, western blot analysis, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and ELISA, were employed to measure relevant biomarkers and cellular responses. Male C57BL/6j mice were treated with atorvastatin to validate in vitro findings. Atorvastatin enhances LDL-C uptake by upregulating LDLR on the cell surface, without causing excess cholesterol accumulation. Additionally, atorvastatin suppresses PCSK9 expression, which is crucial for LDLR degradation. Interestingly, atorvastatin, combined with exogenous LDL-C, impairs glucose-stimulated insulin release (GSIR) but promotes cell proliferation, highlighting a potential mechanism for statin-associated diabetes. Oral administration of atorvastatin in mice reduced plasma PCSK9 and insulin levels, supporting the in vitro findings. These results indicate that while atorvastatin effectively lowers circulating cholesterol, it may adversely affect pancreatic β-cell function by modulating LDLR and LDL-C uptake, thereby increasing the risk of T2D. This study highlights the importance of further research to develop strategies mitigating the diabetogenic effects of statins while maintaining their cardiovascular benefits.

Chronic hyperglycemia impairs mitochondrial function of beta cells. Changes in mitochondrial function preceding a negative glucose effect have not been fully characterized, nor interactions with ketones. To compare effects on beta cell mitochondrial function by short and longer exposures to elevated glucose and interactions with ketones oxygen consumption rate (OCR) was measured in intact clonal beta cells by an OROBOROS and in rat islets by a Seahorse instrument. Proteins (subunits) of mitochondrial complexes (C) were measured by immunoblotting. ATP and ROS were measured in islets. In INS-1 832/13 cells, overnight exposure to 27 vs. 11 mm glucose increased OCR and uncoupled mitochondrial respiration. These effects vanished when prolonging the exposure time of elevated glucose. C1 was decreased after two days of culture with high glucose. Interactions with racemic 5 and 20 mm beta-hydroxybutyrate (BHB) were not detected. In islets, culture overnight at 27 vs.11 mm glucose enhanced basal OCR. No decrease in glucose-induced OCR was seen after prolonging 27 mm glucose for two days. Interactions with 5 mm BHB were not detected. Prolonged exposure to 27 mm glucose enhanced basal ECAR (extracellular acidification rate) and an ECAR response to acute elevation of glucose. C1 and 3 and 4 were decreased after two days of 27 vs. 11 mm glucose. ATP levels were decreased at this time-point and extracellular ROS increased. High glucose time-dependently affects mitochondrial function in clonal beta cells and islets. C1 was uniformly decreased. Interactions with BHB were not detected.

Background: Islet or β-cell transplantation is a therapeutical approach to substitute the insulin-producing cells which are abolished in type 1 diabetes mellitus. The shortage of human islets as well as the complicated and costly isolation process limit the application of these techniques in daily clinical practice. EndoC-βH is a human β-cell line that readily forms aggregates termed pseudoislets, providing an alternative to primary human islets or β-cells.

Methods: EndoC-βH3 cells were seeded and incubated to form pseudoislets. Their insulin secretion was analyzed by ELISA and compared with cell monolayers. Pseudoislets were transplanted into streptozotocin-treated NMRi nu/nu mice. Blood glucose was monitored before and after transplantation and compared with wild types. Grafts were analyzed by immunohistology.

Results: This study shows that EndoC-βH cells are able to form pseudoislets by aggregation, leading to an enhanced glucose stimulated insulin secretion in vitro. These pseudoislets were then successfully transplanted into the livers of diabetic mice and produced insulin in vitro. Blood glucose levels of the streptozocin-treated recipient mice were significantly decreased when compared to pre-transplantation and matched the levels found in control mice.

Conclusion: We suggest pseudoislets aggregated from EndoC-βH cells as a valuable and promising model for islet transplantation research.

Pancreatic islet transplantation is a promising treatment for type 1 diabetes, but the survival and function of transplanted islets are hindered by the loss of extracellular matrix (ECM) during islet isolation and by low oxygenation upon implantation. This study aimed to evaluate the impact of hypoxia on ECM using a cutting-edge imaging approach based on tissue clearing and 3D microscopy. Human and rat islets were cultured under normoxic (O₂ 21%) or hypoxic (O₂ 1%) conditions. Immunofluorescence staining targeting insulin, glucagon, CA9 (a hypoxia marker), ECM proteins (collagen 4, fibronectin, laminin), and E-cadherin (intercellular adhesion protein) was performed on fixed whole islets. The cleared islets were imaged using Light Sheet Fluorescence Microscopy (LSFM) and digitally analyzed. The volumetric analysis of target proteins did not show significant differences in abundance between the experimental groups. However, 3D projections revealed distinct morphological features that differentiated normoxic and hypoxic islets. Under normoxic conditions, ECM could be found throughout the islets. Hypoxic islets exhibited areas of scattered nuclei and central clusters of ECM proteins, indicating central necrosis. E-cadherin was absent in these areas. Our results, demonstrating a diminution of islets' functional mass in hypoxia, align with the functional decline observed in transplanted islets experiencing low oxygenation after grafting. This study provides a methodology combining tissue clearing, multiplex immunofluorescence, Light Sheet Fluorescence Microscopy, and digital image analysis to investigate pancreatic islet morphology. This 3D approach allowed us to highlight ECM organizational changes during hypoxia from a morphological perspective.

Islet amyloid polypeptide (IAPP) is a factor that regulates food intake and is secreted from both pancreatic islets and insulinoma cells. Here, we aimed to evaluate IAPP immunohistochemically in islets or insulinoma cells in association with clinical characteristics. We recruited six insulinoma patients and six body mass index-matched control patients with pancreatic diseases other than insulinoma whose glucose tolerance was confirmed to be normal preoperatively. IAPP and IAPP-insulin double staining were performed on pancreatic surgical specimens. We observed that the IAPP staining level and percentage of IAPP-positive beta cells tended to be lower (p = 0.1699) in the islets of insulinoma patients than in those of control patients, which might represent a novel IAPP expression pattern under persistent hyperinsulinemia and hypoglycemia.