scholarly journals Determination of secretory granule maturation times in pancreatic islet beta-cells by serial block face scanning electron microscopy

2019 ◽  
Author(s):  
A. Rao ◽  
E.L. McBride ◽  
G. Zhang ◽  
H. Xu ◽  
T. Cai ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Beta Cell ◽  
Pancreatic Islets ◽  
Beta Cells ◽  
Secretory Granule ◽  
Secretory Granules ◽  
Pancreatic Islets Of Langerhans ◽  
Beta Cell Volume ◽  
Block Face ◽  
Electron Lucent

AbstractIt is shown how serial block-face electron microscopy (SBEM) of insulin-secreting beta cells in wild-type mouse pancreatic islets of Langerhans can be used to determine maturation times of secretory granules. Although SBEM captures the beta cell structure at a snapshot in time, the observed ultrastructure can be considered representative of a dynamic equilibrium state of the cells since the pancreatic islets are maintained in culture in approximate homeostasis. It is found that 7.2±1.2% (±st. dev.) of the beta cell volume is composed of secretory granule dense-cores exhibiting angular shapes surrounded by wide (typically ≳100 nm) electron-lucent halos. These organelles are identified as mature granules that store insulin for regulated release through the plasma membrane, with a release time of 96±12 hours, as previously obtained from pulsed 35S-radiolabeling of cysteine and methionine. Analysis of beta cell 3D volumes reveals a subpopulation of secretory organelles without electron-lucent halos, identified as immature secretory granules. Another subpopulation of secretory granules is found with thin (typically ≲30 nm) electron-lucent halos, which are attributed to immature granules that are transforming from proinsulin to insulin by action of prohormone convertases. From the volume ratio of proinsulin in the immature granules to insulin in the mature granules, we estimate that the newly formed immature granules remain in morphologically-defined immature states for an average time of 135±14 minutes, and the immature transforming granules for an average time of 130±17 minutes.

Electron microscopy of the pancreatic islets stimulated by insulin antibody

1968 ◽  
Vol 46 (3) ◽  
pp. 407-410 ◽  
Author(s):  
John Logothetopoulos
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Beta Cell ◽  
Pancreatic Islets ◽  
Beta Cells ◽  
Guinea Pigs ◽  
Secretory Granules ◽  
Initial Period ◽  
And Control ◽  
Regressive Changes

Mice were maintained hyperglycemic by daily intraperitoneal injections of immunoglobulins from guinea pigs highly immunized with beef insulin. Hyperglycemic and control mice were killed throughout a period of 3 weeks. Inflammatory infiltrations around the islets were minimal The stimulated beta cells remained heavily degranulated throughout the period of injections. Insignificant changes were seen on the cytomembrane of the beta cell facing the capillary during the initial period of rapid degranulation and thereafter. The density of the rough endoplasmic reticulum increased progressively. The Golgi complexes expanded and became more elaborate. The mitochondria enlarged. Evidence for the continuous elaboration of secretory granules in the Golgi area and for the existence of an immature pregranule in the formation cycle of the secretory granule was obtained in both normal and hyperglycemic animals. Regressive changes in the cells of the islets were absent.

scholarly journals Quick-freeze fixation and freeze-drying of isolated rat pancreatic islets: application to the ultrastructural localization of inorganic phosphate in the pancreatic beta cell.

1981 ◽  
Vol 29 (2) ◽  
pp. 321-325 ◽  
Author(s):  
R W Dudek ◽  
A F Boyne ◽  
N Freinkel
Keyword(s):  
Beta Cell ◽  
Pancreatic Islets ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Inorganic Phosphate ◽  
Freeze Drying ◽  
Secretory Granules ◽  
Pancreatic Beta Cell ◽  
Ultrastructural Localization ◽  
Freeze Fixation

A bounce-free mechanical quick-freeze assembly and a Coulter-Terracio freeze-dry apparatus were successfully coupled to obtain high quality ultrastructural preservation of pancreatic beta cells in a simple and dependable manner. Except for obvious shrinkage spaces, morphological relationships at the tissue, cellular, and subcellular levels were all intact. Beta cell secretory granules demonstrated a dense core surrounded by an electron lucent halo as typically described in specimens after aqueous fixation. Cell membranes and intracellular membranes demonstrated a trilaminar appearance. Golgi apparatus were well preserved. Two clearly defined populations of mitochondria were found. One group of very dark mitochondria had extremely dense matrices in which cristae were barely visible. A second group of mitochondria had light matrices with prominent cristae. The combined quick-freeze fixation and freeze-drying was applied to reevaluate the ultrastructural localization of inorganic phosphate that had been precipitated with lead in the beta cells of pancreatic islets. Accumulation of inorganic phosphate adjacent to the plasma membrane and over the nucleolus of the beta cell in nonstimulated islets was documented with better detail than heretofore possible.

Effects of ciglitazone on the pancreatic islets of C57BL/6J-OB/OB mice: Qualitative electron microscopy of beta cells and quantitative light microscopy of islet surface area and number

1983 ◽  
Vol 41 ◽  
pp. 780-781
Author(s):  
T. Peterson ◽  
G. Sawada ◽  
B. Wyse ◽  
B. Gilchrist ◽  
A. Diani
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Pancreatic Islets ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Islet Cells ◽  
Secretory Granules ◽  
Hypoglycemic Agent ◽  
Quantitative Light Microscopy ◽  
Insulin Storage

The Ob/Ob mouse of the genetic background C57BL/6J is characterized by obesity, hyperglycemia, insulin resistance, and hyperplasia of the pancreatic beta cells. Under electron microscopy, the beta cells from this animal model display degranulation, proliferation of the rough endoplasmic reticulum, and expansion of the Golgi apparatus, all of which are indicative of a stressed pattern of insulin storage and synthesis.Ciglitazone has been reported to function as an oral hypoglycemic agent in the 0b/0b mouse. This depression of plasma glucose was associated with a significant elevation in pancreatic insulin as observed in our laboratory after six weeks of drug treatment (100 mg/kg-day incorporated into Purina Mouse Chow). Pancreatic islets of treated and age-sex matched control C57BL/6J-Ob/Ob mice were then analyzed by ultramorphology and light microscopy to determine the effect of ciglitazone on beta cells. Under electron microscopy, the pancreatic beta cells of treated mice were well granulated with rare abnormalities of the citoplasmic organelles and insulin secretory granules occupied most of the cytoplasm of the islet - cells.

scholarly journals Maturation Times of Pancreatic Beta Cell Secretory Granules Estimated from Serial Block-Face Electron Microscopy

2019 ◽  
Vol 25 (S2) ◽  
pp. 1380-1381
Author(s):  
RD Leapman ◽  
MA Aronova ◽  
A Rao ◽  
EL McBride ◽  
G Zhang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Beta Cell ◽  
Secretory Granules ◽  
Pancreatic Beta Cell ◽  
Block Face

Expression, localization and functional role of small GTPases of the Rab3 family in insulin-secreting cells

1996 ◽  
Vol 109 (9) ◽  
pp. 2265-2273 ◽  
Author(s):  
R. Regazzi ◽  
M. Ravazzola ◽  
M. Iezzi ◽  
J. Lang ◽  
A. Zahraoui ◽  
...  
Keyword(s):  
Pancreatic Islets ◽  
Beta Cells ◽  
Insulin Release ◽  
Cell Lines ◽  
Secretory Granules ◽  
Small Gtpases ◽  
Wild Type ◽  
Gtp Hydrolysis ◽  
Human Pancreatic Islets ◽  
Insulin Secreting Cells

We examined the presence of small molecular mass GTP-binding proteins of the Rab3 family in different insulin-secreting cells. Rab3B and Rab3C were identified by western blotting in rat and in human pancreatic islets, in two rat insulin-secreting cell lines, RINm5F and INS-1, as well as in the hamster cell line HIT-T15. In contrast, Rab3A was detected in rat pancreatic islets as well as in the two insulin-secreting rat cell lines but not in human pancreatic islets and was only barely discernible in HIT-T15 cells. These findings were confirmed by two-dimensional gel electrophoresis followed by GTP-overlay of homogenates of pancreatic islets and of the purified protein. Northern blotting analysis revealed that Rab3D is expressed in the same insulin-secreting cells as Rab3A. Separation of the cells of the rat islets by fluorescence-activated cell sorting demonstrated that Rab3A was exclusively expressed in beta-cells. Rab3A was found to be associated with insulin-containing secretory granules both by immunofluorescence, immunoelectron microscopy and after sucrose density gradient. Overexpression in HIT-T15 cells of a Rab3A mutant deficient in GTP hydrolysis inhibited insulin secretion stimulated by a mixture of nutrients and bombesin. Insulin release triggered by these secretagogues was also slightly decreased by the overexpression of wild-type Rab3A but not by the overexpression of wild-type Rab5A and of a Rab5A mutant deficient in GTP hydrolysis. Finally, we studied the expression in insulin-secreting cells of rabphilin-3A, a putative effector protein that associates with the GTP-bound form of Rab3A. This Rab3A effector was not detectable in any of the cells investigated in the present study. Taken together these results indicate an involvement of Rab3A in the control of insulin release in rat and hamster. In human beta-cells, a different Rab3 isoform but with homologous function may replace Rab3A.

Comparative toxicity of alloxan, N-alkylalloxans and ninhydrin to isolated pancreatic islets in vitro

1997 ◽  
Vol 155 (2) ◽  
pp. 283-293 ◽  
Author(s):  
A Jorns ◽  
R Munday ◽  
M Tiedge ◽  
S Lenzen
Keyword(s):  
Beta Cell ◽  
Pancreatic Islets ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Ultrastructural Changes ◽  
Cell Necrosis ◽  
High Concentrations ◽  
Beta Cell Necrosis

The in vitro toxicity of the diabetogenic agent alloxan as documented by the induction of beta cell necrosis was studied in isolated ob/ob mouse pancreatic islets. The effect of alloxan has been compared with that of a number of N-alkyl alloxan derivatives and with that of the structurally related compound, ninhydrin. Alloxan and its derivatives were selectively toxic to pancreatic beta cells, with other endocrine cells and exocrine parenchymal cells being well preserved, even at high concentration. In contrast, ninhydrin was selectively toxic to pancreatic beta cells only at comparatively low concentration, destroying all islet cell types at high concentrations. The ultrastructural changes induced by all the test compounds in pancreatic beta cells in vitro were very similar to those observed during the development of alloxan diabetes in vivo. The relative toxicity of the various compounds to pancreatic beta cells in vitro was not, however, related to their ability to cause diabetes in vivo. Indeed, the non-diabetogenic substances ninhydrin, N-butylalloxan and N-isobutylalloxan were very much more toxic to isolated islets than the diabetogenic compounds alloxan and N-methylalloxan. These results suggest that the differences in diabetogenicity among alloxan derivatives are not due to intrinsic differences in the susceptibility of the pancreatic beta cells to their toxicity, but may reflect differences in distribution or metabolism. High concentrations of glucose protected islets against the harmful effects of alloxan and its derivatives, but not those of ninhydrin. Low levels of glucose, and non-carbohydrate nutrients, afforded little protection, indicating that the effect of glucose is not due to the production of reducing equivalents within the cell, 3-O-Methylglucose, which protects against alloan diabetes in vivo, did not protect against alloxan toxicity in vitro. Since 3-O-methylglucose is known to prevent uptake of alloxan by pancreatic beta cells, it appears that uptake of alloxan by the cell is not a prerequisite for the induction of beta cell necrosis.

scholarly journals Endothelial and beta cell composite aggregates for improved function of a bioartificial pancreas encapsulation device

2018 ◽  
Vol 41 (3) ◽  
pp. 152-159 ◽  
Author(s):  
Katarzyna Skrzypek ◽  
Yazmin Brito Barrera ◽  
Thomas Groth ◽  
Dimitrios Stamatialis
Keyword(s):  
Endothelial Cells ◽  
Insulin Secretion ◽  
Beta Cell ◽  
Pancreatic Islets ◽  
Beta Cells ◽  
Cell Function ◽  
Umbilical Vein ◽  
Human Umbilical Vein ◽  
Min6 Cells ◽  
Umbilical Vein Endothelial Cells

Introduction: Encapsulation of pancreatic islets or beta cells is a promising strategy for treatment of type 1 diabetes by providing an immune isolated environment and allowing for transplantation in a different location than the liver. However, islets used for encapsulation often show lower functionality due to the damaging of islet endothelial cells during the isolation procedure. Factors produced by endothelial cells have great impact on beta cell insulin secretion. Therefore, mutual signaling between endothelial cells and beta cells should be considered for the development of encapsulation systems to achieve high insulin secretion and maintain beta cell viability. Here, we investigate whether co-culture of beta cells with endothelial cells could improve beta cell function within encapsulation devices. Materials and methods: Mouse insulinoma MIN6 cells and human umbilical vein endothelial cells were used for creating composite aggregates on agarose microwell platform. The composite aggregates were encapsulated within flat poly(ether sulfone)/polyvinylpyrrolidone device. Their functionality was assessed by glucose-induced insulin secretion test and compared to non-encapsulated free-floating aggregates. Results: We created composite aggregates of 80–100 µm in diameter, closely mimicking pancreatic islets. Upon glucose stimulation, their insulin secretion is improved in comparison to aggregates consisting of only MIN6 cells. Moreover, the composite aggregates encapsulated within a device secrete more insulin than aggregates consisting of only MIN6 cells. Conclusion: Composite aggregates of MIN6 cells with human umbilical vein endothelial cells have improved insulin secretion in comparison to MIN6 aggregates showing that the interaction of beta cell and endothelial cell is crucial for a functional encapsulation system.

scholarly journals PCSK9 affects expression of key surface proteins in human pancreatic beta cells through intra- and extracellular regulatory circuits

2021 ◽  
Author(s):  
kevin Saitoski ◽  
Maria Ryaboshapkina ◽  
Ghaith Hamza ◽  
Andrew F Jarnuczak ◽  
claire berthault ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Beta Cell ◽  
Pancreatic Islets ◽  
Cell Line ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Pancreatic Beta Cell ◽  
Loss Of Function ◽  
Beta Cell Line ◽  
Gain And Loss

Aims/hypothesis: Proprotein convertase subtilisin/kexin 9 (PCSK9) is involved in the degradation of LDLR. However, PCSK9 can target other proteins in a cell-type specific manner. While PCSK9 has been detected in pancreatic islets, its expression in insulin-producing pancreatic beta cells is debated. Herein, we studied PCSK9 expression, regulation and function in the human pancreatic beta cell line EndoC-βH1. Methods: We assessed PCSK9 expression in mouse and human pancreatic islets, and in the pancreatic beta cell line EndoC-βH1. We also studied PCSK9 regulation by cholesterol, lipoproteins, Mevastatin, and by SREBPs transcription factors. To evaluate PCSK9 function in pancreatic beta cells, we performed PCSK9 gain-and loss-of-function experiments in EndoC-βH1 using siPCSK9 or recombinant PCSK9 treatments, respectively. Results: We demonstrate that PCSK9 is expressed and secreted by pancreatic beta cells. In EndoC-βH1 cells, PCSK9 expression is regulated by cholesterol and by SREBPs transcription factors. Importantly, PCSK9 knockdown results in multiple transcriptome, proteome and secretome deregulations and impaired insulin secretion. By gain- and loss-of- function experiments, we observed that PCSK9 regulates the expression levels of LDLR and VLDLR through an extracellular mechanism while CD36, PD-L1 and HLA-ABC are regulated through an intracellular mechanism. Conclusions/interpretation: Collectively, these results highlight PCSK9 as an important regulator of CD36, PD-L1 and HLA-ABC cell surface expression in pancreatic beta cells. Data availability: RNA-seq data have been deposited to GEO database with accession number GSE182016. Mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the following identifiers: PXD027921, PXD027911 and PXD027913.

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