α-catenin isoforms are regulated by glucose and involved in regulating insulin secretion in rat clonal β-cell models

The recent finding that β-catenin levels play an important rate-limiting role in processes regulating insulin secretion lead us to investigate whether its binding partner α-catenin also plays a role in this process. We find that levels of both α-E-catenin and α-N-catenin are rapidly up-regulated as levels of glucose are increased in rat clonal β-cell models INS-1E and INS-832/3. Lowering in levels of either α-catenin isoform using siRNA resulted in significant increases in glucose stimulated insulin secretion (GSIS) and this effect was attenuated when β-catenin levels were lowered indicating these proteins have opposing effects on insulin release. This effect of α-catenin knockdown on GSIS was not due to increases in insulin expression but was associated with increases in calcium influx into cells. Moreover, simultaneous depletion of α-E catenin and α-N catenin decreased the actin polymerisation to a similar degree as latrunculin treatment and inhibition of ARP 2/3 mediated actin branching with CK666 attenuated the α-catenin depletion effect on GSIS. This suggests α-catenin mediated actin remodelling may be involved in the regulation of insulin secretion. Together this indicates that α-catenin and β-catenin can play opposing roles in regulating insulin secretion, with some degree of functional redundancy in roles of α-E-catenin and α-N-catenin. The finding that, at least in β-cell models, the levels of each can be regulated in the longer term by glucose also provides a potential mechanism by which sustained changes in glucose levels might impact on the magnitude of GSIS.

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A role for PAK1 mediated phosphorylation of β-catenin Ser552 in the regulation of insulin secretion

Biochemical Journal ◽

10.1042/bcj20200862 ◽

2021 ◽

Author(s):

Brie Sorrenson ◽

Waruni C Dissanayake ◽

Fengyun Hu ◽

Kate L Lee ◽

Peter R Shepherd

Keyword(s):

Insulin Secretion ◽

Hormonal Regulation ◽

Cell Signalling ◽

Cell Models ◽

Β Cells ◽

Β Cell ◽

Glucose Levels ◽

Phosphorylation Event ◽

A Cell ◽

Glucose Stimulated Insulin Secretion

The presence of adherens junctions and the associated protein β-catenin are requirements for the development of glucose stimulated insulin secretion (GSIS) in β-cells. Evidence indicates that modulation of β-catenin function in response to changes in glucose levels can modulate the levels of insulin secretion from β-cells but the role of β-catenin phosphorylation in this process has not been established. We find that a Ser552Ala version of β-catenin attenuates glucose stimulated insulin secretion indicating a functional role for Ser552 phosphorylation of β-catenin in insulin secretion. This is associated with alterations F/G actin ratio but not transcriptional activity of β-catenin. Both glucose and GLP-1 stimulated phosphorylation of the serine 552 residue on β-catenin. We investigated the possibility that an EPAC-PAK1 pathway might be involved in this phosphorylation event. We find that reduction in PAK1 levels using siRNA attenuates both glucose and GLP-1 stimulated phosphorylation of β-catenin Ser552 and the effects of these on insulin secretion in β-cell models. Further, both the EPAC inhibitor ESI-09 and the PAK1 inhibitor IPA3 do the same in both β-cell models and mouse islets. Together this identifies phosphorylation of β-catenin at Ser552 as part of a cell signalling mechanism linking nutrient and hormonal regulation of β-catenin to modulation of insulin secretory capacity of β-cells and indicates this phosphorylation event is regulated downstream of EPAC and PAK1 in β-cells.

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Identification of a pathway by which glucose regulates β-catenin signalling via the cAMP/protein kinase A pathway in β-cell models

Biochemical Journal ◽

10.1042/bj20121454 ◽

2013 ◽

Vol 449 (3) ◽

pp. 803-811 ◽

Cited By ~ 15

Author(s):

Emmanuelle Cognard ◽

Coralie G. Dargaville ◽

Deborah L. Hay ◽

Peter R. Shepherd

Keyword(s):

Insulin Secretion ◽

Protein Kinase ◽

Protein Kinase A ◽

Cell Function ◽

Regulation Of Gene Expression ◽

Cell Models ◽

Β Cells ◽

Β Cell ◽

Glucose Levels ◽

Kinase A

Pancreatic β-cells are highly responsive to changes in glucose, but the mechanisms involved are only partially understood. There is increasing evidence that the β-catenin signalling pathway plays an important role in regulating β-cell function, but the mechanisms regulating β-catenin signalling in these cells is not well understood. In the present study we show that β-catenin levels and downstream signalling are regulated by changes in glucose levels in INS-1E and β-TC6-F7 β-cell models. We found a glucose-dependent increase in levels of β-catenin in the cytoplasm and nucleus of INS-1E cells. Expression of cyclin D1 also increased with glucose and required the presence of β-catenin. This was associated with an increase in phosphorylation of β-catenin on Ser552, which is known to stabilize the molecule and increase its transcriptional activity. In a search for possible signalling intermediates we found forskolin and cell-permeable cAMP analogues recapitulated the glucose effects, suggesting a role for cAMP and PKA (cAMP-dependent protein kinase/protein kinase A) downstream of glucose. Furthermore, glucose caused sustained increases in cAMP. Two different inhibitors of adenylate cyclase and PKA signalling blocked the effects of glucose, whereas siRNA (small interfering RNA) knockdown of PKA blocked the effects of glucose on β-catenin signalling. Finally, reducing β-catenin levels with either siRNA or pyrvinium impaired glucose- and KCl-stimulated insulin secretion. Taken together the results of the present study define a pathway by which changes in glucose levels can regulate β-catenin using a mechanism which involves cAMP production and the activation of PKA. This identifies a pathway that may be important in glucose-dependent regulation of gene expression and insulin secretion in β-cells.

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Loss of Liver Kinase B1 (LKB1) in Beta Cells Enhances Glucose-stimulated Insulin Secretion Despite Profound Mitochondrial Defects

Journal of Biological Chemistry ◽

10.1074/jbc.m115.639237 ◽

2015 ◽

Vol 290 (34) ◽

pp. 20934-20946 ◽

Cited By ~ 22

Author(s):

Avital Swisa ◽

Zvi Granot ◽

Natalia Tamarina ◽

Sophie Sayers ◽

Nabeel Bardeesy ◽

...

Keyword(s):

Insulin Secretion ◽

Cell Polarity ◽

Cell Biology ◽

Calcium Influx ◽

Cell Mass ◽

Negative Regulator ◽

Β Cells ◽

Β Cell ◽

Liver Kinase B1 ◽

Glucose Stimulated Insulin Secretion

The tumor suppressor liver kinase B1 (LKB1) is an important regulator of pancreatic β cell biology. LKB1-dependent phosphorylation of distinct AMPK (adenosine monophosphate-activated protein kinase) family members determines proper β cell polarity and restricts β cell size, total β cell mass, and glucose-stimulated insulin secretion (GSIS). However, the full spectrum of LKB1 effects and the mechanisms involved in the secretory phenotype remain incompletely understood. We report here that in the absence of LKB1 in β cells, GSIS is dramatically and persistently improved. The enhancement is seen both in vivo and in vitro and cannot be explained by altered cell polarity, increased β cell number, or increased insulin content. Increased secretion does require membrane depolarization and calcium influx but appears to rely mostly on a distal step in the secretion pathway. Surprisingly, enhanced GSIS is seen despite profound defects in mitochondrial structure and function in LKB1-deficient β cells, expected to greatly diminish insulin secretion via the classic triggering pathway. Thus LKB1 is essential for mitochondrial homeostasis in β cells and in parallel is a powerful negative regulator of insulin secretion. This study shows that β cells can be manipulated to enhance GSIS to supra-normal levels even in the face of defective mitochondria and without deterioration over months.

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Ontology of the apelinergic system in mouse pancreas during pregnancy and relationship with β-cell mass

Scientific Reports ◽

10.1038/s41598-021-94725-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Brenda Strutt ◽

Sandra Szlapinski ◽

Thineesha Gnaneswaran ◽

Sarah Donegan ◽

Jessica Hill ◽

...

Keyword(s):

Cell Proliferation ◽

Insulin Secretion ◽

Glucose Transporter ◽

Cell Mass ◽

Elevated Serum ◽

Pancreatic Β Cell ◽

Β Cells ◽

Β Cell ◽

Glucose Transporter 2 ◽

Glucose Stimulated Insulin Secretion

AbstractThe apelin receptor (Aplnr) and its ligands, Apelin and Apela, contribute to metabolic control. The insulin resistance associated with pregnancy is accommodated by an expansion of pancreatic β-cell mass (BCM) and increased insulin secretion, involving the proliferation of insulin-expressing, glucose transporter 2-low (Ins+Glut2LO) progenitor cells. We examined changes in the apelinergic system during normal mouse pregnancy and in pregnancies complicated by glucose intolerance with reduced BCM. Expression of Aplnr, Apelin and Apela was quantified in Ins+Glut2LO cells isolated from mouse pancreata and found to be significantly higher than in mature β-cells by DNA microarray and qPCR. Apelin was localized to most β-cells by immunohistochemistry although Aplnr was predominantly associated with Ins+Glut2LO cells. Aplnr-staining cells increased three- to four-fold during pregnancy being maximal at gestational days (GD) 9–12 but were significantly reduced in glucose intolerant mice. Apelin-13 increased β-cell proliferation in isolated mouse islets and INS1E cells, but not glucose-stimulated insulin secretion. Glucose intolerant pregnant mice had significantly elevated serum Apelin levels at GD 9 associated with an increased presence of placental IL-6. Placental expression of the apelinergic axis remained unaltered, however. Results show that the apelinergic system is highly expressed in pancreatic β-cell progenitors and may contribute to β-cell proliferation in pregnancy.

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Abstract 294: The A2b Adenosine Receptor and Newly Identified Opposing Effects of cAMP on Insulin Secretion

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.32.suppl_1.a294 ◽

2012 ◽

Vol 32 (suppl_1) ◽

Author(s):

Hillary A Johnston-Cox ◽

Barbara Corkey ◽

Katya Ravid

Keyword(s):

Insulin Secretion ◽

Adenosine Receptor ◽

Underlying Mechanism ◽

Short Exposure ◽

A2b Adenosine Receptor ◽

Downstream Effects ◽

Glucose Challenge ◽

Short And Long Term ◽

Glucose Stimulated Insulin Secretion ◽

Opposing Effects

The A2b adenosine receptor (A2bAR) is a G-protein coupled receptor that, upon binding of adenosine, activates adenylyl cyclase and mediates downstream effects through secondary messengers, including cyclic 3’5’ AMP (cAMP) and Ca ++ . We have previously demonstrated that A2bAR knockout (KO) KO mice, post-high fat diet (HFD) develop a type 2 diabetic (T2D) phenotype, evidenced by elevated plasma insulin and glucose. Pancreatic islets from A2bAR KO mice demonstrated insulin hypersecretion post-4 weeks HFD, and high glucose challenge. To further understand the underlying mechanism, we focused on the contribution of the pancreatic A2bAR to this phenomnena. cAMP has been demonstrated to be a significant amplifier of glucose-stimulated insulin secretion. Through the use of A2bAR KO islets and diet-induced stress, we identified a new dual role for cAMP in mediating insulin secretion, dependent on cAMP level and duration. Short exposure to elevated cAMP indeed causes insulin hypersecretion. cAMP has, however, also a downregulating effect on the expression of GLUT2 mRNA and protein, which has the potential to inhibit insulin secretion. Thus, A2bAR short and long-term signaling in the pancreas play an important role in insulin homeostasis.

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Bioactive Phytochemicals Isolated from Akebia quinata Enhances Glucose-Stimulated Insulin Secretion by Inducing PDX-1

Plants ◽

10.3390/plants9091087 ◽

2020 ◽

Vol 9 (9) ◽

pp. 1087

Author(s):

Dahae Lee ◽

Jin Su Lee ◽

Jurdas Sezirahiga ◽

Hak Cheol Kwon ◽

Dae Sik Jang ◽

...

Keyword(s):

Insulin Secretion ◽

Cell Function ◽

Experimental Studies ◽

Pancreatic Β Cell ◽

Β Cell ◽

Etoh Extract ◽

Chemical Structures ◽

Β Cell Function ◽

Glucose Stimulated Insulin Secretion ◽

Phosphoinositide 3 Kinase

Chocolate vine (Akebia quinata) is consumed as a fruit and is also used in traditional medicine. In order to identify the bioactive components of A. quinata, a phytosterol glucoside stigmasterol-3-O-β-d-glucoside (1), three triterpenoids maslinic acid (2), scutellaric acid (3), and hederagenin (4), and three triterpenoidal saponins akebia saponin PA (5), hederacoside C (6), and hederacolchiside F (7) were isolated from a 70% EtOH extract of the fruits of A. quinata (AKQU). The chemical structures of isolates 1–7 were determined by analyzing the 1D and 2D nuclear magnetic resonance (NMR) spectroscopic data. Here, we evaluated the effects of AKQU and compounds 1–7 on insulin secretion using the INS-1 rat pancreatic β-cell line. Glucose-stimulated insulin secretion (GSIS) was evaluated in INS-1 cells using the GSIS assay. The expression levels of the proteins related to pancreatic β-cell function were detected by Western blotting. Among the isolates, stigmasterol-3-O-β-d-glucoside (1) exhibited strong GSIS activity and triggered the overexpression of pancreas/duodenum homeobox protein-1 (PDX-1), which is implicated in the regulation of pancreatic β-cell survival and function. Moreover, isolate 1 markedly induced the expression of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), insulin receptor substrate-2 (IRS-2), phosphoinositide 3-kinase (PI3K), and Akt, which regulate the transcription of PDX-1. The results of our experimental studies indicated that stigmasterol-3-O-β-d-glucoside (1) isolated from the fruits of A. quinata can potentially enhance insulin secretion, and might alleviate the reduction in GSIS during the development of T2DM.

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L-Arginine prevents cereblon-mediated ubiquitination of glucokinase and stimulates glucose-6-phosphate production in pancreatic β-cells

Communications Biology ◽

10.1038/s42003-020-01226-3 ◽

2020 ◽

Vol 3 (1) ◽

Author(s):

Jaeyong Cho ◽

Yukio Horikawa ◽

Mayumi Enya ◽

Jun Takeda ◽

Yoichi Imai ◽

...

Keyword(s):

Insulin Secretion ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Β Cells ◽

Β Cell ◽

Pancreatic Β Cells ◽

Direct Stimulation ◽

Glucose Ratio ◽

Glucose Stimulated Insulin Secretion

Abstract We sought to determine a mechanism by which L-arginine increases glucose-stimulated insulin secretion (GSIS) in β-cells by finding a protein with affinity to L-arginine using arginine-immobilized magnetic nanobeads technology. Glucokinase (GCK), the key regulator of GSIS and a disease-causing gene of maturity-onset diabetes of the young type 2 (MODY2), was found to bind L-arginine. L-Arginine stimulated production of glucose-6-phosphate (G6P) and induced insulin secretion. We analyzed glucokinase mutants and identified three glutamate residues that mediate binding to L-arginine. One MODY2 patient with GCKE442* demonstrated lower C-peptide-to-glucose ratio after arginine administration. In β-cell line, GCKE442* reduced L-arginine-induced insulin secretion compared with GCKWT. In addition, we elucidated that the binding of arginine protects glucokinase from degradation by E3 ubiquitin ligase cereblon mediated ubiquitination. We conclude that L-arginine induces insulin secretion by increasing G6P production by glucokinase through direct stimulation and by prevention of degradation.

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The adaptor protein APPL2 controls glucose-stimulated insulin secretion via F-actin remodeling in pancreatic β-cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2016997117 ◽

2020 ◽

Vol 117 (45) ◽

pp. 28307-28315

Author(s):

Baile Wang ◽

Huige Lin ◽

Xiaomu Li ◽

Wenqi Lu ◽

Jae Bum Kim ◽

...

Keyword(s):

Insulin Secretion ◽

Pancreatic Islets ◽

Adaptor Protein ◽

Hek293 Cells ◽

Β Cells ◽

Β Cell ◽

Pancreatic Β Cells ◽

Actin Remodeling ◽

Actin Depolymerization ◽

Glucose Stimulated Insulin Secretion

Filamentous actin (F-actin) cytoskeletal remodeling is critical for glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells, and its dysregulation causes type 2 diabetes. The adaptor protein APPL1 promotes first-phase GSIS by up-regulating solubleN-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein expression. However, whether APPL2 (a close homology of APPL1 with the same domain organization) plays a role in β-cell functions is unknown. Here, we show that APPL2 enhances GSIS by promoting F-actin remodeling via the small GTPase Rac1 in pancreatic β-cells. β-cell specific abrogation of APPL2 impaired GSIS, leading to glucose intolerance in mice. APPL2 deficiency largely abolished glucose-induced first- and second-phase insulin secretion in pancreatic islets. Real-time live-cell imaging and phalloidin staining revealed that APPL2 deficiency abolished glucose-induced F-actin depolymerization in pancreatic islets. Likewise, knockdown of APPL2 expression impaired glucose-stimulated F-actin depolymerization and subsequent insulin secretion in INS-1E cells, which were attributable to the impairment of Ras-related C3 botulinum toxin substrate 1 (Rac1) activation. Treatment with the F-actin depolymerization chemical compounds or overexpression of gelsolin (a F-actin remodeling protein) rescued APPL2 deficiency-induced defective GSIS. In addition, APPL2 interacted with Rac GTPase activating protein 1 (RacGAP1) in a glucose-dependent manner via the bin/amphiphysin/rvs-pleckstrin homology (BAR-PH) domain of APPL2 in INS-1E cells and HEK293 cells. Concomitant knockdown of RacGAP1 expression reverted APPL2 deficiency-induced defective GSIS, F-actin remodeling, and Rac1 activation in INS-1E cells. Our data indicate that APPL2 interacts with RacGAP1 and suppresses its negative action on Rac1 activity and F-actin depolymerization thereby enhancing GSIS in pancreatic β-cells.

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Human EndoC-βH1 β-cells form pseudoislets with improved glucose sensitivity and enhanced GLP-1 signaling in the presence of islet-derived endothelial cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00272.2017 ◽

2018 ◽

Vol 314 (5) ◽

pp. E512-E521 ◽

Cited By ~ 1

Author(s):

Michael G. Spelios ◽

Lauren A. Afinowicz ◽

Regine C. Tipon ◽

Eitan M. Akirav

Keyword(s):

Endothelial Cells ◽

Insulin Secretion ◽

Cell Biology ◽

Three Dimensional ◽

Cell Types ◽

Human Islets ◽

Glucose Sensing ◽

Β Cells ◽

Β Cell ◽

Glucose Levels

Three-dimensional (3D) pseudoislets (PIs) can be used for the study of insulin-producing β-cells in free-floating islet-like structures similar to that of primary islets. Previously, we demonstrated the ability of islet-derived endothelial cells (iECs) to induce PIs using murine insulinomas, where PI formation enhanced insulin production and glucose responsiveness. In this report, we examined the ability of iECs to spontaneously induce the formation of free-floating 3D PIs using the EndoC-βH1 human β-cell line murine MS1 iEC. Within 14 days, the coculturing of both cell types produced fully humanized EndoC-βH1 PIs with little to no contaminating murine iECs. The size and shape of these PIs were similar to primary human islets. iEC-induced PIs demonstrated reduced dysregulated insulin release under low glucose levels and higher insulin secretion in response to high glucose and exendin-4 [a glucagon-like peptide-1 (GLP-1) analog] compared with monolayer cells cultured alone. Interestingly, iEC-PIs were also better at glucose sensing in the presence of extendin-4 compared with PIs generated on a low-adhesion surface plate in the absence of iECs and showed an overall improvement in cell viability. iEC-induced PIs exhibited increased expression of key genes involved in glucose transport, glucose sensing, β-cell differentiation, and insulin processing, with a concomitant decrease in glucagon mRNA expression. The enhanced responsiveness to exendin-4 was associated with increased protein expression of GLP-1 receptor and phosphokinase A. This rapid coculture system provides an unlimited number of human PIs with improved insulin secretion and GLP-1 responsiveness for the study of β-cell biology.

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Investigation of Ultrasound-Mediated Intracellular Ca2+ Oscillations in HIT-T15 Pancreatic β-Cell Line

Cells ◽

10.3390/cells9051129 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1129

Author(s):

Chi Woo Yoon ◽

Nan Sook Lee ◽

Kweon Mo Koo ◽

Sunho Moon ◽

Kyosuk Goo ◽

...

Keyword(s):

Insulin Secretion ◽

Cell Line ◽

Purinergic Signaling ◽

Underlying Mechanism ◽

Resting Cells ◽

Pancreatic Β Cell ◽

Pulsed Ultrasound ◽

Β Cell ◽

Secretion Pathways ◽

Glucose Stimulated Insulin Secretion

In glucose-stimulated insulin secretion (GSIS) of pancreatic β-cells, the rise of free cytosolic Ca2+ concentration through voltage-gated calcium channels (VGCCs) triggers the exocytosis of insulin-containing granules. Recently, mechanically induced insulin secretion pathways were also reported, which utilize free cytosolic Ca2+ ions as a direct regulator of exocytosis. In this study, we aimed to investigate intracellular Ca2+ responses on the HIT-T15 pancreatic β-cell line upon low-intensity pulsed ultrasound (LIPUS) stimulation and found that ultrasound induces two distinct types of intracellular Ca2+ oscillation, fast-irregular and slow-periodic, from otherwise resting cells. Both Ca2+ patterns depend on the purinergic signaling activated by the rise of extracellular ATP or ADP concentration upon ultrasound stimulation, which facilitates the release through mechanosensitive hemichannels on the plasma membrane. Further study demonstrated that two subtypes of purinergic receptors, P2X and P2Y, are working in a competitive manner depending on the level of glucose in the cell media. The findings can serve as an essential groundwork providing an underlying mechanism for the development of a new therapeutic approach for diabetic conditions with further validation.

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