scholarly journals Sequential in vivo labeling of insulin secretory granule pools in INS-SNAP transgenic pigs

2021 ◽  
Vol 118 (37) ◽  
pp. e2107665118
Author(s):  
Elisabeth Kemter ◽  
Andreas Müller ◽  
Martin Neukam ◽  
Anna Ivanova ◽  
Nikolai Klymiuk ◽  
...  
Keyword(s):  
Secretory Granule ◽  
Cell Function ◽  
Ex Vivo ◽  
Secretory Granules ◽  
Pig Model ◽  
Transgenic Pigs ◽  
Glucose Levels ◽  
Molecular Features ◽  
Insulin Secretory Granule

β cells produce, store, and secrete insulin upon elevated blood glucose levels. Insulin secretion is a highly regulated process. The probability for insulin secretory granules to undergo fusion with the plasma membrane or being degraded is correlated with their age. However, the molecular features and stimuli connected to this behavior have not yet been fully understood. Furthermore, our understanding of β cell function is mostly derived from studies of ex vivo isolated islets in rodent models. To overcome this translational gap and study insulin secretory granule turnover in vivo, we have generated a transgenic pig model with the SNAP-tag fused to insulin. We demonstrate the correct targeting and processing of the tagged insulin and normal glycemic control of the pig model. Furthermore, we show specific single- and dual-color granular labeling of in vivo–labeled pig pancreas. This model may provide unprecedented insights into the in vivo insulin secretory granule behavior in an animal close to humans.

scholarly journals Sequential in vivo labeling of insulin secretory granule pools in INS-SNAP transgenic pigs

2021 ◽  
Author(s):  
Elisabeth Kemter ◽  
Andreas Müller ◽  
Martin Neukam ◽  
Anna Ivanova ◽  
Nikolai Klymiuk ◽  
...  
Keyword(s):  
Secretory Granule ◽  
Cell Function ◽  
Ex Vivo ◽  
Secretory Granules ◽  
Pig Model ◽  
Transgenic Pigs ◽  
Glucose Levels ◽  
Molecular Features ◽  
Insulin Secretory Granule

β-cells produce, store and secrete insulin upon elevated blood glucose levels. Insulin secretion is a highly regulated process. The probability for insulin secretory granules to undergo fusion with the plasma membrane or being degraded is correlated with their age. However, the molecular features and stimuli connected to this behavior have not yet been fully understood. Furthermore, our understanding of β-cell function is mostly derived from studies of ex vivo isolated islets and/or rodent models. To overcome this translational gap and study insulin secretory granule turnover in vivo, we have generated a transgenic pig model with the SNAP-tag fused to insulin. We demonstrate the correct targeting and processing of the tagged insulin and normal glycemic control of the pig model. Furthermore, we show specific single- and dual-color granular labeling of in vivo labeled pig pancreas. This model may provide unprecedented insights into the in vivo insulin secretory granule behavior in an animal close to humans.

scholarly journals A fluorescent timer reporter enables sorting of insulin secretory granules by age

2020 ◽  
Vol 295 (27) ◽  
pp. 8901-8911 ◽  
Author(s):  
Belinda Yau ◽  
Lori Hays ◽  
Cassandra Liang ◽  
D. Ross Laybutt ◽  
Helen E. Thomas ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Cell Function ◽  
Islet Cells ◽  
Ex Vivo ◽  
Secretory Granules ◽  
Β Cells ◽  
Β Cell ◽  
Short Period ◽  
Insulin Granule

Within the pancreatic β-cells, insulin secretory granules (SGs) exist in functionally distinct pools, displaying variations in motility as well as docking and fusion capability. Current therapies that increase insulin secretion do not consider the existence of these distinct SG pools. Accordingly, these approaches are effective only for a short period, with a worsening of glycemia associated with continued decline in β-cell function. Insulin granule age is underappreciated as a determinant for why an insulin granule is selected for secretion and may explain why newly synthesized insulin is preferentially secreted from β-cells. Here, using a novel fluorescent timer protein, we aimed to investigate the preferential secretion model of insulin secretion and identify how granule aging is affected by variation in the β-cell environment, such as hyperglycemia. We demonstrate the use of a fluorescent timer construct, syncollin-dsRedE5TIMER, which changes its fluorescence from green to red over 18 h, in both microscopy and fluorescence-assisted organelle-sorting techniques. We confirm that the SG-targeting construct localizes to insulin granules in β-cells and does not interfere with normal insulin SG behavior. We visualize insulin SG aging behavior in MIN6 and INS1 β-cell lines and in primary C57BL/6J mouse and nondiabetic human islet cells. Finally, we separated young and old insulin SGs, revealing that preferential secretion of younger granules occurs in glucose-stimulated insulin secretion. We also show that SG population age is modulated by the β-cell environment in vivo in the db/db mouse islets and ex vivo in C57BL/6J islets exposed to different glucose environments.

scholarly journals CONCOMITANT SYNTHESIS OF MEMBRANE PROTEIN AND EXPORTABLE PROTEIN OF THE SECRETORY GRANULE IN RAT PAROTID GLAND

1971 ◽  
Vol 50 (1) ◽  
pp. 187-200 ◽  
Author(s):  
Abraham Amsterdam ◽  
Michael Schramm ◽  
Itzhak Ohad ◽  
Yoram Salomon ◽  
Zvi Selinger
Keyword(s):  
Amino Acids ◽  
Secretory Granule ◽  
De Novo ◽  
Secretory Granules ◽  
Specific Radioactivity ◽  
Staining Intensity ◽  
Granule Membrane ◽  
Rat Parotid ◽  
Cell Fractions

After enzyme secretion the membrane of the secretory granule, which had been fused to the cell membrane, was resorbed into the cell. Experiments were therefore carried out to test whether formation of new secretory granules involves reutilization of the resorbed membrane or synthesis of a new membrane, de novo, from amino acids. Incorporation of amino acids-14C into proteins of various cell fractions was measured in vivo, 30, 120, and. 300 min after labeling. At all times the specific radioactivity of the secretory granule membrane was about equal to that of the granule's exportable content. At 120 and 300 min the specific radioactivity of the granule membrane and of the granule content was much higher than that of any other subcellular fraction. It is therefore concluded that the protein of the membrane is synthesized de novo concomitantly with the exportable protein. The proteins of the granule membrane could be distinguished from those of the granule content by gel electrophoresis. All major bands were labeled proportionately to their staining intensity. The amino acid composition of the secretory granule membrane was markedly different from that of the granule's content and also from that of the mitochondrial membrane. The granule membrane showed a high proline content, 30 moles/100 moles amino acids. The analyses show that the radioactivity of the granule membrane is indeed inherent in its proteins and is not due to contamination by other fractions. The possibility is considered that the exportable protein leaves the endoplasmic reticulum already enveloped by the newly synthesized membrane.

TNFR25 Expression on CD4+CD25+ T Cells: Down Modulation of Regulatory Activity.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3165-3165
Author(s):  
Vadim Deyev ◽  
Melinda Roskos ◽  
Robert B. Levy ◽  
Eckhard R. Podack
Keyword(s):  
T Cells ◽  
Cell Function ◽  
Ex Vivo ◽  
Cell Activity ◽  
Treg Cells ◽  
Regulatory Activity ◽  
T Regulatory Cell ◽  
Tnf Receptor Family ◽  
Receptor Family

Abstract TNFR25 (“DR3”) is a member of the TNF receptor family that is expressed by activated CD4+ and CD8+ T cells. To determine if activated CD4+CD25+ T cells also expressed this TNFR family molecule, B6 CD4+CD25+ T cells were stimulated with anti-CD3/CD28 coated beads (kind gift of Dr. B. Blazar, U. Minn.) and expanded for 3–4 days. TNFR25 expression was readily detected on CD4+CD25+ FoxP3+ T cells. Since other members of the TNF receptor family (GITR, OX40, 4–1BB) are known to influence T regulatory cell function, we investigated whether TNFR25 signaling can regulate CD4+CD25+ T cell activity. TNFR25 triggering in B6-wt T regulatory CD4+CD25+ cells with the recombinant TNFR25 ligand TL1A or agonistic anti-TNFR25 antibody (4C12) resulted in reduction of their ability to suppress anti-CD3 induced ex-vivo proliferation of CD4+CD25− cells. 4C12 mediated TNFR25 signaling also reduced B6-wt Treg mediated inhibition of peptide induced proliferation of OVA-specific B6 CD8+ (OT-I) cells. To further investigate a role for TNFR25 in Treg cell regulation, TNFR25 (full length) transgenic mice were generated and bred onto the BL/6 background. CD4+CD25+ cells from these TNFR25 tg mice were found to possess diminished T regulatory activity in vitro as determined by their diminished inability to regulate proliferation by B6-wt CD4+ and OT-I CD8+ T cells. To assess their in vivo regulatory activity, B6-wt and B6 TNFR25 tg Treg cells were examined for their ability to inhibit graft vs. host disease (GVHD) following allogeneic MHC class I/II mismatched BMT. In contrast to B6-wt Treg cells, TNFR25 tg Treg cells exhibited significantly diminished ability to regulate the onset of GVHD in vivo as assessed by weight loss and clinical symptoms. Using agonistic antibody, stimulation of TNFR25 on transgenic Treg cells was also found to effectively remove the ex-vivo regulatory activity expressed by this population. To exclude any possible direct co-stimulatory effects of 4C12 antibody on the responding proliferating cells, CD4+CD25−T cells from TNFR25 dominant negative transgenic mice were employed. 4C12 mab again abolished Treg cell inhibitory activity. The effect of TNFR25 agonists on T reg cell activity in vivo is being further investigated in both mouse models of GVHD and IBD diseases. Initial observations administering 4C12 post-allogeneic BMT together with B6-wt Treg cells indicate a reduction in their ability to regulate GVHD. In total, these studies identify TNFR25 as a new potential target for augmenting CD4+ and CD8+ responses by concomitant direct co-stimulation of effecter cells and inhibition of T regulatory cell function.

scholarly journals Clinical indices of in vivo biocompatibility: The role of ex vivo cell function studies and effluent markers in peritoneal dialysis patients

2003 ◽  
Vol 64 ◽  
pp. S84-S93 ◽  
Author(s):  
Ruth Mackenzie ◽  
Clifford J. Holmes ◽  
Suzanne Jones ◽  
John D. Williams ◽  
Nicholas Topley
Keyword(s):  
Peritoneal Dialysis ◽  
Cell Function ◽  
Ex Vivo ◽  
Dialysis Patients ◽  
Clinical Indices

scholarly journals The Novel TRPA1 Antagonist BI01305834 Inhibits Ovalbumin-Induced Bronchoconstriction in Guinea Pigs

2020 ◽  
Author(s):  
Mariska van den Berg ◽  
Susan Nijboer - Brinksma ◽  
Sophie Bos ◽  
Maarten van den Berge ◽  
David Lamb ◽  
...  
Keyword(s):  
Pilot Study ◽  
Guinea Pig ◽  
Guinea Pigs ◽  
Ex Vivo ◽  
Optimal Dose ◽  
Pig Model ◽  
The Novel ◽  
Airway Narrowing ◽  
Guinea Pig Model

Abstract Background: We hypothesized that TRPA1 channels contribute to airway hyperresponsiveness (AHR) and inflammation in asthma. We evaluated the efficacy of the novel TRPA1 antagonist BI01305834 in a guinea pig model of asthma. Methods: First a pilot study was performed in a guinea pig model of allergic asthma to find the optimal dose of BI01305834. Next, the effect of BI01305834 on AHR to inhaled histamine after the early and late asthmatic reaction (EAR and LAR), magnitude of EAR and LAR and airway inflammation was assessed. Precision-cut lung slices and trachea strips were used to investigate the bronchoprotective and bronchodilating effect of BI01305834. Statistical evaluation of differences of in vivo data was performed using a Mann-Whitney U test or One-way nonparametric Kruskal-Wallis ANOVA, for ex vivo data One- or Two-way ANOVA was used, all with Dunnett’s post-hoc test where appropriate.Results: A dose of 1 mg/kg BI01305834 was selected based on AHR and exposure data in blood samples from the pilot study. In the subsequent study 1 mg/kg BI01305834 inhibited AHR after the EAR, and the development of EAR and LAR elicited by ovalbumin in ovalbumin-sensitized guinea pigs. BI01305834 did not inhibit allergen-induced total and differential cells in the lavage fluid and interleukin-13 gene expression in lung homogenates. Furthermore, BI01305834 was able to inhibit allergen and histamine-induced airway narrowing in guinea pig lung slices, without affecting histamine release, and reverse allergen-induced bronchoconstriction in guinea pig trachea strips.Conclusions: TRPA1 inhibition protects against AHR and the EAR and LAR in vivo and allergen and histamine-induced airway narrowing ex vivo, and reverses allergen-induced bronchoconstriction, independently of inflammation. This effect was partially dependent upon histamine, suggesting a neuronal and possible non-neuronal role for TRPA1 in allergen-induced bronchoconstriction.

scholarly journals 3D FIB-SEM reconstruction of microtubule–organelle interaction in whole primary mouse β cells

2020 ◽  
Vol 220 (2) ◽  
Author(s):  
Andreas Müller ◽  
Deborah Schmidt ◽  
C. Shan Xu ◽  
Song Pang ◽  
Joyson Verner D’Costa ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cell Function ◽  
Secretory Granules ◽  
Detailed Knowledge ◽  
Β Cells ◽  
Microtubule Organization ◽  
Supportive Role ◽  
Primary Mouse ◽  
Microtubule Networks ◽  
Insulin Secretory Granule

Microtubules play a major role in intracellular trafficking of vesicles in endocrine cells. Detailed knowledge of microtubule organization and their relation to other cell constituents is crucial for understanding cell function. However, their role in insulin transport and secretion is under debate. Here, we use FIB-SEM to image islet β cells in their entirety with unprecedented resolution. We reconstruct mitochondria, Golgi apparati, centrioles, insulin secretory granules, and microtubules of seven β cells, and generate a comprehensive spatial map of microtubule–organelle interactions. We find that microtubules form nonradial networks that are predominantly not connected to either centrioles or endomembranes. Microtubule number and length, but not microtubule polymer density, vary with glucose stimulation. Furthermore, insulin secretory granules are enriched near the plasma membrane, where they associate with microtubules. In summary, we provide the first 3D reconstructions of complete microtubule networks in primary mammalian cells together with evidence regarding their importance for insulin secretory granule positioning and thus their supportive role in insulin secretion.

scholarly journals Molecular characterization of clonal human renal forming cells

2020 ◽  
Author(s):  
Cohen-Zontag Osnat ◽  
Gershon Rotem ◽  
Harari-Steinberg Orit ◽  
Kanter Itamar ◽  
Omer Dorit ◽  
...  
Keyword(s):  
Cell Function ◽  
Epithelial Mesenchymal Transition ◽  
Bmp Signaling ◽  
Mesenchymal Transition ◽  
Clonal Cell ◽  
Human Adult ◽  
Molecular Features ◽  
Adult Kidney ◽  
Clonal Cultures

AbstractThe adult kidney replaces its parenchyma in vivo in steady state and during regeneration by segment-specific clonal cell proliferation.To understand human adult kidney clonal cell growth, we derived tissue from human nephrectomies and performed limiting dilution to establish genuine clonal cultures from one single cell.Clonal efficiency of the human kidney was x%. Remarkably, a single renal cell could give rise to up to 3.3*10(6) cells. Phenotypically, two types of clonal cultures were apparent; a stably proliferating cuboidal epithelial-like appearing (EL) and a rapidly proliferating fibroblast-like appearing (FL). RNA sequencing of all clonal cultures separated FL from EL cultures according to proximal-distal/collecting renal epithelial tubular identity, respectively. Moreover, distinct molecular features in respect to cell-cycle, epithelial-mesenchymal transition, oxidative phosphorylation, BMP signaling pathway and cell surface markers were observed for each clone type. Surprisingly, clonal expansion (>3 months) was sustained in EL clones harboring markers of mature kidney epithelia (high CD24, CDH1, EpCAM, EMA) in contrast to de-differentiated FL clones (high NCAM1, serpine1), which showed fast lineage amplification and exhausted in a few weeks.Thus, the human adult kidney harbors progenitor cell function in which segment identity and the level of epithelial differentiation dictate clonal characteristics.

Effects of Ammonium Perfluorooctanoate on Leydig-Cell Function: In Vitro, in Vivo, and ex Vivo Studies

1995 ◽  
Vol 134 (1) ◽  
pp. 18-25 ◽  
Author(s):  
L.B. Biegel ◽  
R.C.M. Liu ◽  
M.E. Hurtt ◽  
J.C. Cook
Keyword(s):  
Leydig Cell ◽  
Cell Function ◽  
Ex Vivo ◽  
Leydig Cell Function

scholarly journals The insulin-secretory-granule carboxypeptidase H. Purification and demonstration of involvement in proinsulin processing

1987 ◽  
Vol 245 (2) ◽  
pp. 575-582 ◽  
Author(s):  
H W Davidson ◽  
J C Hutton
Keyword(s):  
Secretory Granule ◽  
Gel Electrophoresis ◽  
Secretory Granules ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Carboxypeptidase B ◽  
Carboxypeptidase H ◽  
Insulin Secretory Granule

A carboxypeptidase B-like enzyme was detected in the soluble fraction of purified insulin secretory granules, and implicated in insulin biosynthesis. To investigate the role of this activity further, we purified the enzyme from rat insulinoma tissue by gel-filtration chromatography and affinity elution from p-aminobenzoyl-arginine. A yield of 42%, with a purification factor of 674 over the homogenate, was achieved. Analysis of the purified carboxypeptidase by SDS/polyacrylamide-gel electrophoresis under either reducing or non-reducing conditions showed it to be a monomeric protein of apparent Mr 55,000. The preparation was also homogeneous by high-performance gel-filtration chromatography. The enzyme bound to concanavalin A, showing it to be a glycoprotein. Amino acid analysis or chemical deglycosylation and SDS/polyacrylamide-gel electrophoresis indicated a protein Mr of 50,000, suggesting a carbohydrate content of approx. 9% by weight. The purified enzyme was able to remove basic amino acids from the C-terminus of proinsulin tryptic peptides to generate insulin, but did not further degrade the mature hormone. It was inhibited by EDTA, 1,10-phenanthroline and guanidinoethylmercaptosuccinic acid, and stimulated 5-fold by CoCl2. The pH optimum of the conversion of diarginyl-insulin into insulin was in the range 5-6, with little activity above pH 6.5. Activity was also expressed towards a dansylated tripeptide substrate (dansyl-phenylalanyl-leucyl-arginine; Km = 17.5 microM), and had a pH optimum of 5.5. These properties are indistinguishable from those of the activity located in secretory granules, and are compatible with the intragranular environment. The insulin-secretory-granule carboxypeptidase shared several properties of carboxypeptidase H from bovine adrenal medulla and pituitary. We propose that the carboxypeptidase that we purified is the pancreatic isoenzyme of carboxypeptidase H (crino carboxypeptidase B; EC 3.4.17.10), and is involved in the biosynthesis of insulin in the pancreatic beta-cell.

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