scholarly journals Purification of age-distinct insulin secretory granules through antigen restriction using the CLIP-tag

2020 ◽  
Author(s):  
Martin Neukam ◽  
Katharina Ganß ◽  
Jovana Vasiljević ◽  
Johannes Broichhagen ◽  
Kai Johnsson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Insulin Secretion ◽  
Beta Cells ◽  
Solid Phase ◽  
Secretory Granules ◽  
Islet Beta Cells ◽  
Temporal Variables ◽  
Pancreatic Islet Beta Cells ◽  
Insulin Granules ◽  
Insulin Granule

AbstractPancreatic islet beta cells employ secretory granules for the storage and glucose-stimulated release of the hormone insulin. The competence of an insulin granule for exocytosis depends on spatial and temporal variables such as its proximity to the plasma membrane as well as its age, with newly-generated granules being preferentially released. The molecular underpinnings for the control of these variables remain largely unknown and their uncovering is of high relevance for the study of diabetes, which results from deficient insulin secretion. However, we still lack a comprehensive view about the molecular composition of the insulin granules and how this may change over their lifetime. Here we report a strategy for the background-free purification of insulin secretory granules of distinct age from insulinoma INS-1 cells. We show that utilization of an immuno-based affinity approach for pulse-chase labeled insulin secretory granules, produces a highly enriched granular fraction. Our approach precludes typical contaminants from the solid phase and may be designed to purify secretory granules of a distinct age.

scholarly journals Glucose-induced ERM protein activation and translocation regulates insulin secretion

2010 ◽  
Vol 299 (5) ◽  
pp. E772-E785 ◽  
Author(s):  
James P. Lopez ◽  
Jerrold R. Turner ◽  
Louis H. Philipson
Keyword(s):  
Plasma Membrane ◽  
Insulin Secretion ◽  
Dominant Negative ◽  
Scaffolding Protein ◽  
Actin Binding ◽  
Cell Periphery ◽  
Dependent Manner ◽  
Erm Proteins ◽  
Insulin Granules ◽  
Insulin Granule

A key step in regulating insulin secretion is insulin granule trafficking to the plasma membrane. Using live-cell time-lapse confocal microscopy, we observed a dynamic association of insulin granules with filamentous actin and PIP2-enriched structures. We found that the scaffolding protein family ERM, comprising ezrin, radixin, and moesin, are expressed in β-cells and target both F-actin and PIP2. Furthermore, ERM proteins are activated via phosphorylation in a glucose- and calcium-dependent manner. This activation leads to a translocation of the ERM proteins to sites on the cell periphery enriched in insulin granules, the exocyst complex docking protein Exo70, and lipid rafts. ERM scaffolding proteins also participate in insulin granule trafficking and docking to the plasma membrane. Overexpression of a truncated dominant-negative ezrin construct that lacks the ERM F-actin binding domain leads to a reduction in insulin granules near the plasma membrane and impaired secretion. Conversely, overexpression of a constitutively active ezrin results in more granules near the cell periphery and an enhancement of insulin secretion. Diabetic mouse islets contain less active ERM, suggestive of a novel mechanism whereby impairment of insulin granule trafficking to the membrane through a complex containing F-actin, PIP2, Exo70, and ERM proteins contributes to defective insulin secretion.

scholarly journals Quantitative morphometric studies of pancreatic islets obtained from tolbutamide-treated rats.

1986 ◽  
Vol 34 (9) ◽  
pp. 1195-1200 ◽  
Author(s):  
D E Yorde ◽  
R K Kalkhoff
Keyword(s):  
Pancreatic Islets ◽  
Beta Cells ◽  
Secretory Granules ◽  
Total Cell ◽  
Islet Beta Cells ◽  
Physiological Conditions ◽  
Pancreatic Islet Beta Cells ◽  
Granule Density ◽  
Treatment Data ◽  
Computerized System

We have developed a computerized system for quantitative morphometric analysis of the number and position of secretory granules and organelles in pancreatic islet beta cells following tolbutamide treatment. Data from animals injected with tolbutamide for 1, 2, and 3 days were compared to tissues obtained from untreated control animals. Pancreatic islets removed by a collagenase technique were perfused with an appropriate medium to restore a basal state. After fixation and embedding, thick sections of beta cells were viewed by electron microscopy. Morphometric studies of randomly selected or serially cut cells were performed with computer programs for digitization, quantify, rotational, and perspective display. Tolbutamide treatment resulted in graded granule depletion which was maximal at 72 hr relative to control animals. Reduced granule density was associated with significant reduction in total cell area or cytoplasmic area, but was without effect on nuclear size. Since granule depletion improved visualization of subcellular structures, this will enable us to pursue studies of exocytosis under a variety of physiological conditions.

scholarly journals The PI(4)P phosphatase Sac2 controls insulin granule docking and release

2019 ◽  
Vol 218 (11) ◽  
pp. 3714-3729 ◽  
Author(s):  
Phuoc My Nguyen ◽  
Nikhil R. Gandasi ◽  
Beichen Xie ◽  
Sari Sugahara ◽  
Yingke Xu ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Plasma Membrane ◽  
Insulin Secretion ◽  
Dependent Manner ◽  
Impaired Insulin Secretion ◽  
Insulin Granules ◽  
Impaired Insulin ◽  
Insulin Granule ◽  
Phosphatidylinositol 4

Insulin granule biogenesis involves transport to, and stable docking at, the plasma membrane before priming and fusion. Defects in this pathway result in impaired insulin secretion and are a hallmark of type 2 diabetes. We now show that the phosphatidylinositol 4-phosphate phosphatase Sac2 localizes to insulin granules in a substrate-dependent manner and that loss of Sac2 results in impaired insulin secretion. Sac2 operates upstream of granule docking, since loss of Sac2 prevented granule tethering to the plasma membrane and resulted in both reduced granule density and number of exocytic events. Sac2 levels correlated positively with the number of docked granules and exocytic events in clonal β cells and with insulin secretion in human pancreatic islets, and Sac2 expression was reduced in islets from type 2 diabetic subjects. Taken together, we identified a phosphoinositide switch on the surface on insulin granules that is required for stable granule docking at the plasma membrane and impaired in human type 2 diabetes.

scholarly journals Age matters: Grading granule secretion in beta cells

2020 ◽  
Vol 295 (27) ◽  
pp. 8912-8913 ◽  
Author(s):  
Eleni Georgiadou ◽  
Guy A. Rutter
Keyword(s):  
Beta Cells ◽  
Secretory Granules ◽  
Islet Beta Cells ◽  
Quantitative Evidence ◽  
Glucose Levels ◽  
Rapid Release ◽  
Pancreatic Islet Beta Cells ◽  
Age Dependent ◽  
Granule Secretion ◽  
Organelle Sorting

Insulin is stored in secretory granules to facilitate rapid release in response to rising glucose levels, but the mechanisms by which these granules are identified and prioritized for secretion remains unclear. Using a fluorescent timer and flow cytometry–assisted organelle sorting, Yau et al. develop an elegant approach to assess insulin secretion as a function of granule age in pancreatic islet beta cells. Their findings supply quantitative evidence supporting the age-dependent release of different granule pools and confirm earlier models of preferential release of younger granules.

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