scholarly journals Adequate connexin-mediated coupling is required for proper insulin production.

1995 ◽  
Vol 131 (6) ◽  
pp. 1561-1572 ◽  
Author(s):  
C Vozzi ◽  
S Ullrich ◽  
A Charollais ◽  
J Philippe ◽  
L Orci ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Gap Junctions ◽  
Beta Cells ◽  
Stable Transfection ◽  
Insulin Production ◽  
Wild Type ◽  
Transfected Cells ◽  
Insulin Producing Cells ◽  
And Storage ◽  
Rat Insulinoma

To assess whether connexin (Cx) expression contributes to insulin secretion, we have investigated normal and tumoral insulin-producing cells for connexins, gap junctions, and coupling. We have found that the glucose-sensitive cells of pancreatic islets and of a rat insulinoma are functionally coupled by gap junctions made of Cx43. In contrast, cells of several lines secreting insulin abnormally do not express Cx43, gap junctions, and coupling. After correction of these defects by stable transfection of Cx43 cDNA, cells expressing modest levels of Cx43 and coupling, as observed in native beta-cells, showed an expression of the insulin gene and an insulin content that were markedly elevated, compared with those observed in both wild-type (uncoupled) cells and in transfected cells overexpressing Cx43. These findings indicate that adequate levels of Cx-mediated coupling are required for proper insulin production and storage.

scholarly journals Pancreatic beta-cells expressing the Arg64 variant of the beta(3)-adrenergic receptor exhibit abnormal insulin secretory activity

2001 ◽  
Vol 27 (2) ◽  
pp. 133-144 ◽  
Author(s):  
R Perfetti ◽  
H Hui ◽  
K Chamie ◽  
S Binder ◽  
M Seibert ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Beta Cells ◽  
Western Blotting ◽  
Adrenergic Receptor ◽  
Pancreatic Beta Cells ◽  
Secretory Activity ◽  
Host Cells ◽  
Human Pancreas ◽  
Dependent Manner ◽  
Wild Type

The Arg64 beta(3)-adrenergic receptor (beta(3)AR) variant is associated with an earlier age of onset of diabetes and lower levels of insulin secretion in humans. The aims of this study were to investigate whether beta(3)AR is expressed by islet cells, if receptor binding affects insulin secretion and, finally, if the beta(3)AR Arg64 variant induces abnormal insulin secretory activity. Human pancreas extracts were subjected to RT-PCR, Western blotting and immunostaining analyses. DNA sequencing and Western blotting demonstrated that the beta(3)AR gene is transcribed and translated in the human pancreas; immunostaining showed that it is expressed by the islets of Langerhans. Cultured rat beta-cells responded to human beta(3)AR agonists in a dose- and time-dependent manner. Transfection of cultured rat beta-cells with the wild-type human beta(3)AR produced an increased baseline and ligand-dependent insulin secretion compared with parental cells. On the other hand, cells transfected with the Arg64 variant of the beta(3)AR secreted less insulin, both spontaneously and after exposure to human beta(3)AR agonists. Furthermore, while transfection with the wild-type beta(3)AR preserved the glucose-dependent secretion of insulin, expression of the variant receptor rendered the host cells significantly less responsive to glucose. In summary, cells express the beta(3)AR, and its activation contributes to the regulation of insulin secretion. These findings may help explain the low levels of insulin secretion in response to an i.v. glucose tolerance test observed in humans carrying the Arg64 polymorphism.

Dysregulated lncRNAs Regulate Human Umbilical Cord Mesenchymal Stem Cells Differentiation into Insulin-Producing Cells by Forming a Regulatory Network with mRNAs

2021 ◽  
Author(s):  
Tianqin Xie ◽  
Qiming Huang ◽  
Qiulang Huang ◽  
Haixia Zeng ◽  
Jianping Liu
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Insulin Secretion ◽  
Signaling Pathway ◽  
Beta Cells ◽  
Umbilical Cord ◽  
Pancreatic Beta Cells ◽  
Differentially Expressed ◽  
Human Umbilical Cord ◽  
Insulin Producing Cells

Abstract ObjectiveIn recent years, cell therapy has become a new research direction in the treatment of diabetes. However, the underlying molecular mechanisms of mesenchymal stem cells (MSCs) participate in such treatment has not been clarified. MethodsIn this study, human umbilical cord mesenchymal stem cells (HUC-MSCs) isolated from newborns were progressively induced into insulin-producing cells (IPCs) using small molecules. HUC-MSCs (S0) and four induced stage (S1-S4) samples were prepared. We then performed transcriptome sequencing experiments to obtain the dynamic expression profiles of both mRNAs and long noncoding RNAs (lncRNAs). ResultsWe found that the number of differentially expressed lncRNAs and mRNAs showed a decreasing trend during differentiation. Gene Ontology (GO) analysis showed that the target genes of differentially expressed lncRNAs were associated with translation, cell adhesion, and cell connection. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the NF-KB signaling pathway, MAPK signaling pathway, HIPPO signaling pathway, PI3K-Akt signaling pathway, and p53 signaling pathway were enriched in these differentially expressed lncRNA-targeting genes. We also found that the coexpression of the lncRNA: CTBP1-AS2 with the PROX1, and the lncRNAs AC009014.3 and GS1-72M22.1 with the mRNA JARID2 was related to the development of pancreatic beta cells. Moreover, the coexpression of the lncRNAs :XLOC_ 050969, LINC00883, XLOC_050981, XLOC_050925, MAP3K14- AS1, RP11-148K1.12, and CTD2020K17.3 with p53, regulated insulin secretion by pancreatic beta cells.ConclusionThis research revealed that HUC-MSCs combined with small molecule compounds were successfully induced into IPCs. Differentially expressed lncRNAs may regulate the insulin secretion of pancreatic beta cells by regulating multiple signaling pathways. The lncRNAs: AC009014.3,Gs1-72m21.1 and CTBP1-AS2 may be involved in the development of pancreatic beta cells, and the lncRNAs: XLOC_050969, LINC00883, XLOC_050981, XLOC_050925, MAP3K14-AS1, RP11-148K1.12, and CTD2020K17.3 may be involved in regulating the insulin secretion of pancreatic beta cells, thus providing a lncRNA catalog for future research regarding the mechanism of the transdifferentiation of HUC-MSCs into IPCs. It also provides a new theoretical basis for the transplantation of insulin-producing cells into diabetic patients in the future.

Nonrandom distribution of gap junctions between pancreatic beta-cells

1980 ◽  
Vol 238 (3) ◽  
pp. C114-C119 ◽  
Author(s):  
P. Meda ◽  
J. F. Denef ◽  
A. Perrelet ◽  
L. Orci
Keyword(s):  
Insulin Secretion ◽  
Gap Junctions ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Freeze Fracture ◽  
Membrane Area ◽  
Nonrandom Distribution ◽  
Isolated Rat Islets ◽  
Rat Islets Of Langerhans ◽  
Stimulation Of

The numerical and spatial distribution of gap junctions between insulin-containing cells (beta-cells) under resting and stimulated conditions of insulin secretion were quantitatively analyzed in freeze-fracture replicas of isolated rat islets of Langerhans. The results show that the beta-cells located at the periphery of the islet have twice as many gap junctions per unit membrane area as the beta-cells situated in the islet center. In both locations, gap junctions assumed a nonrandom clustering on the beta-cell membranes. During stimulation of insulin secretion, the gap junctions were found increased between the central and between the peripheral beta-cells. The degree of their clustering was also modified. The latter change depended both on the location of the gap junctions in the islet and on the type of stimulation used (high glucose or glibenclamide).

scholarly journals Connexin26 deafness associated mutations show altered permeability to large cationic molecules

2008 ◽  
Vol 295 (4) ◽  
pp. C966-C974 ◽  
Author(s):  
Gülistan Meşe ◽  
Virginijus Valiunas ◽  
Peter R. Brink ◽  
Thomas W. White
Keyword(s):  
Gap Junctions ◽  
Voltage Clamp ◽  
Intercellular Communication ◽  
Essential Role ◽  
Lucifer Yellow ◽  
Cell Voltage ◽  
Wild Type ◽  
Transfected Cells ◽  
Large Molecules ◽  
Ionic Coupling

Intercellular communication is important for cochlear homeostasis because connexin26 (Cx26) mutations are the leading cause of hereditary deafness. Gap junctions formed by different connexins have unique selectivity to large molecules, so compensating for the loss of one isoform can be challenging in the case of disease causing mutations. We compared the properties of Cx26 mutants T8M and N206S with wild-type channels in transfected cells using dual whole cell voltage clamp and dye flux experiments. Wild-type and mutant channels demonstrated comparable ionic coupling, and their average unitary conductance was ∼106 and ∼60 pS in 120 mM K+-aspartate− and TEA+-aspartate− solution, respectively, documenting their equivalent permeability to K+ and TEA+. Comparison of cAMP, Lucifer Yellow (LY), and ethidium bromide (EtBr) transfer revealed differences in selectivity for larger anionic and cationic tracers. cAMP and LY permeability to wild-type and mutant channels was similar, whereas the transfer of EtBr through mutant channels was greatly reduced compared with wild-type junctions. Altered permeability of Cx26 to large cationic molecules suggests an essential role for biochemical coupling in cochlear homeostasis.

scholarly journals Tetracycline-regulated secretion of human insulin in a transfected non-endocrine cell line

2003 ◽  
Vol 30 (3) ◽  
pp. 331-346 ◽  
Author(s):  
KT Scougall ◽  
CA Maltin ◽  
JA Shaw
Keyword(s):  
Insulin Secretion ◽  
Cell Line ◽  
Human Insulin ◽  
Endocrine Cell ◽  
Therapeutic Potential ◽  
Ex Vivo ◽  
Host Cells ◽  
Cleavage Sites ◽  
Wild Type ◽  
Transfected Cells

Long-term constitutive secretion of insulin by implantation of ex vivo transfected cells such as fibroblasts or myoblasts or in situ by intramuscular injection of naked plasmid DNA provides a potential approach to gene therapy for diabetes mellitus. A mechanism for regulating insulin secretion will be necessary to realize the therapeutic potential of this approach. A second obstacle is the inability of non-endocrine host cells to fully process proinsulin. Therefore, alteration of the wild-type cDNA will be necessary to achieve processing of proinsulin by endogenous endoproteases within these cells. The cDNAs for beta-galactosidase (beta), human wild-type proinsulin (hppI1) and a mutated construct (hppI4), in which the dibasic PC2 and PC3 cleavage sites had been altered to form furin cleavage sites, were sub-cloned into four vectors (pCR3, pVR1012, pIRES, pTRE), including a tetracycline responsive plasmid (pTRE) that requires co-transfection with another plasmid encoding a transactivator (pTet-off) for transgene expression. Transient transfection of the COS-7 fibroblast cell line with these constructs was performed using DEAE-dextran and liposomes. Analysis of vector efficiencies revealed that pTRE/pTet-off>pIRES>pCR3>pVR1012. Further analysis demonstrated total pro/insulin secretion of 2.33 ng/10(6) cells/24 h with > or =25% processed to insulin in hppI-1.pTRE/pTet-off-transfected cells compared with 0.39 ng/10(6) cells/24 h and >70% processing in hppI-4.pTRE/pTet-off-transfected cells. In co-transfection studies with pTRE-hppI1/pTet-off and pTRE-hppI4/pTet-off constructs, pro/insulin secretion was inhibited to 65-66% and 36-38% of control (100%) in the presence of 0.01 and 0.1 microg/ml tetracycline respectively over a 24-h incubation period. Furthermore, reversal of tetracycline inhibition was demonstrated for pTRE-hppI1/pTet-off- and pTRE-hppI4/pTet-off-transfected cells. After a 48-h incubation with 1.0 microg/ml tetracycline, total pro/insulin levels were 10 and 14% compared with untreated cells respectively. On tetracycline removal, total proinsulin levels increased and were equivalent to untreated groups 72 h later. In conclusion, regulation of fully processed human insulin secretion has been achieved in a transiently transfected non-endocrine cell line.

scholarly journals Diffusion of metabolites across gap junctions mediates metabolic coordination of β-islet cells

2020 ◽  
Author(s):  
Vishnu P. Rao ◽  
Megan A. Rizzo
Keyword(s):  
Insulin Secretion ◽  
Gap Junctions ◽  
Beta Cells ◽  
Calcium Influx ◽  
Islet Cells ◽  
Metabolic Coupling ◽  
Heterogeneous Expression ◽  
Low Glucose ◽  
Junctional Activity ◽  
Gap Junctional

AbstractLoss of oscillatory insulin secretion is an early marker of type II diabetes. In an individual beta-cell, insulin secretion is triggered by glucose metabolism, which leads to membrane depolarization and calcium influx. Islet β-cells display coordinated secretion; however, it is unclear how the heterogeneous population of insulin-secreting beta cells coordinate their response to glucose. The mechanisms underlying both electrical and calcium synchronicity are well explored. Even so, the mechanism governing metabolic coordination is unclear given key glycolytic enzymes’ heterogeneous expression. To understand how islet cells coordinate their metabolic activity, a microfluidic applicator delivered glucose stimulation to spatially defined areas of isolated mouse islets. We measured metabolic responses using NADH/NADPH (or NAD(P)H) autofluorescence and calcium changes using Fluo-4 fluorescence. Glucose stimulated a rise in NAD(P)H even in islet areas unexposed to the treatment, suggesting metabolic coordination. A gap junction inhibitor blocked the coordinated NAD(P)H rise in the low-glucose areas. Additionally, metabolic communication did not occur in immortalized β-cell clusters known to lack gap junctions, demonstrating their importance for metabolic coupling. Metabolic communication also preceded glucose-stimulated rises in intracellular calcium. Further, pharmacological blockade of calcium influx did not disrupt NAD(P)H rises in untreated regions. These data suggest that metabolic coordination between islet beta-cells relies on gap-junctional activity and precedes synchronous electrical and calcium activity.

scholarly journals The Transcription Factor COUP-TFII Is Negatively Regulated by Insulin and Glucose via Foxo1- and ChREBP-Controlled Pathways

2008 ◽  
Vol 28 (21) ◽  
pp. 6568-6579 ◽  
Author(s):  
Anaïs Perilhou ◽  
Cécile Tourrel-Cuzin ◽  
Ilham Kharroubi ◽  
Carole Henique ◽  
Véronique Fauveau ◽  
...  
Keyword(s):  
Gene Expression ◽  
Insulin Secretion ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Feedback Loop ◽  
Ex Vivo ◽  
Insulin Production ◽  
Glucose Effect ◽  
Positive Feedback Loop

ABSTRACT COUP-TFII has an important role in regulating metabolism in vivo. We showed this previously by deleting COUP-TFII from pancreatic beta cells in heterozygous mutant mice, which led to abnormal insulin secretion. Here, we report that COUP-TFII expression is reduced in the pancreas and liver of mice refed with a carbohydrate-rich diet and in the pancreas and liver of hyperinsulinemic and hyperglycemic mice. In pancreatic beta cells, COUP-TFII gene expression is repressed by secreted insulin in response to glucose through Foxo1 signaling. Ex vivo COUP-TFII reduces insulin production and secretion. Our results suggest that beta cell insulin secretion is under the control of an autocrine positive feedback loop by alleviating COUP-TFII repression. In hepatocytes, both insulin, through Foxo1, and high glucose concentrations repress COUP-TFII expression. We demonstrate that this negative glucose effect involves ChREBP expression. We propose that COUP-TFII acts in a coordinate fashion to control insulin secretion and glucose metabolism.

scholarly journals FSTL3 Neutralizing Antibodies Enhance Glucose-Responsive Insulin Secretion In Dysfunctional Male Mouse And Human Islets

Endocrinology ◽  
2021 ◽  
Author(s):  
Melissa L Brown ◽  
Alexa Lopez ◽  
Nolan Meyer ◽  
Alden Richter ◽  
Thomas B Thompson
Keyword(s):  
Insulin Secretion ◽  
Beta Cell ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Neutralizing Antibodies ◽  
Cell Number ◽  
Human Islets ◽  
In Vitro Assays ◽  
Insulin Production ◽  
Wide Range

Abstract Diabetes is caused by insufficient insulin production from pancreatic beta cells or insufficient insulin action leading to an inability to control blood glucose. While a wide range of treatments exist to alleviate the symptoms of diabetes, therapies addressing the root cause of diabetes through replacing lost beta cells with functional cells remain an active pursuit. We previously demonstrated that genetic deletion of Fstl3, a critical regulator of activin activity, enhanced beta cell number and glucose-responsive insulin production. These observations suggested the hypothesis that FSTL3 neutralization could be used to therapeutically enhance beta cell number and function in humans. To pursue this possibility, we developed an FSTL3 neutralizing antibody, FP-101, and characterized its ability to prevent or disrupt FSTL3 from complexing with activin or related ligands. This antibody was selective for FSTL3 relative to the closely related follistatin thereby reducing the chance for off-target effects. In vitro assays with FP-101 and activin revealed that FP-101-mediated neutralization of FSTL3 can enhance both insulin secretion and glucose responsiveness to non-functional mouse and human islets under conditions that model diabetes. Thus, FSTL3 neutralization may provide a novel therapeutic strategy for treating diabetes through repairing dysfunctional beta cells.

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