Publications by authors named "Francis C Lynn"

45 Publications

Recapitulating pancreatic cell-cell interactions through bioengineering approaches: the momentous role of non-epithelial cells for diabetes cell therapy.

Cell Mol Life Sci 2021 Dec 6;78(23):7107-7132. Epub 2021 Oct 6.

Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.

Over the past few years, extensive efforts have been made to generate in-vitro pancreatic micro-tissue, for disease modeling or cell replacement approaches in pancreatic related diseases such as diabetes mellitus. To obtain these goals, a closer look at the diverse cells participating in pancreatic development is necessary. Five major non-epithelial pancreatic (pN-Epi) cell populations namely, pancreatic endothelium, mesothelium, neural crests, pericytes, and stellate cells exist in pancreas throughout its development, and they are hypothesized to be endogenous inducers of the development. In this review, we discuss different pN-Epi cells migrating to and existing within the pancreas and their diverse effects on pancreatic epithelium during organ development mediated via associated signaling pathways, soluble factors or mechanical cell-cell interactions. In-vivo and in-vitro experiments, with a focus on N-Epi cells' impact on pancreas endocrine development, have also been considered. Pluripotent stem cell technology and multicellular three-dimensional organoids as new approaches to generate pancreatic micro-tissues have also been discussed. Main challenges for reaching a detailed understanding of the role of pN-Epi cells in pancreas development in utilizing for in-vitro recapitulation have been summarized. Finally, various novel and innovative large-scale bioengineering approaches which may help to recapitulate cell-cell interactions and are crucial for generation of large-scale in-vitro multicellular pancreatic micro-tissues, are discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00018-021-03951-2DOI Listing
December 2021

Premature termination codon readthrough upregulates progranulin expression and improves lysosomal function in preclinical models of GRN deficiency.

Mol Neurodegener 2020 03 16;15(1):21. Epub 2020 Mar 16.

Division of Neurology, University of British Columbia, Vancouver, British Columbia, Canada.

Background: Frontotemporal lobar degeneration (FTLD) is a devastating and progressive disorder, and a common cause of early onset dementia. Progranulin (PGRN) haploinsufficiency due to autosomal dominant mutations in the progranulin gene (GRN) is an important cause of FTLD (FTLD-GRN), and nearly a quarter of these genetic cases are due to a nonsense mutation. Premature termination codons (PTC) can be therapeutically targeted by compounds allowing readthrough, and aminoglycoside antibiotics are known to be potent PTC readthrough drugs. Restoring endogenous PGRN through PTC readthrough has not previously been explored as a therapeutic intervention in FTLD.

Methods: We studied whether the aminoglycoside G418 could increase PGRN expression in HEK293 and human induced pluripotent stem cell (hiPSC)-derived neurons bearing the heterozygous S116X, R418X, and R493X pathogenic GRN nonsense mutations. We further tested a novel substituted phthalimide PTC readthrough enhancer in combination with G418 in our cellular models. We next generated a homozygous R493X knock-in hiPSC isogenic line (R493X KI), assessing whether combination treatment in hiPSC-derived neurons and astrocytes could increase PGRN and ameliorate lysosomal dysfunction relevant to FTLD-GRN. To provide in vivo proof-of-concept of our approach, we measured brain PGRN after intracerebroventricular administration of G418 in mice expressing the V5-tagged GRN nonsense mutation R493X.

Results: The R418X and R493X mutant GRN cell lines responded to PTC readthrough with G418, and treatments increased PGRN levels in R493X KI hiPSC-derived neurons and astrocytes. Combining G418 with a PTC readthrough enhancer increased PGRN levels over G418 treatment alone in vitro. PGRN deficiency has been shown to impair lysosomal function, and the mature form of the lysosomal protease cathepsin D is overexpressed in R493X KI neurons. Increasing PGRN through G418-mediated PTC readthrough normalized this abnormal lysosomal phenotype in R493X KI neuronal cultures. A single intracerebroventricular injection of G418 induced GRN PTC readthrough in 6-week-old AAV-GRN-R493X-V5 mice.

Conclusions: Taken together, our findings suggest that PTC readthrough may be a potential therapeutic strategy for FTLD caused by GRN nonsense mutations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13024-020-00369-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075020PMC
March 2020

Super-resolution microscopy compatible fluorescent probes reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics.

Nat Commun 2020 01 24;11(1):467. Epub 2020 Jan 24.

Institute of Metabolism and Systems Research (IMSR), and Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham, Birmingham, UK.

The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection or the use of probes that stimulate receptor activation. Herein, we present LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label. LUXendin645 produces intense and specific membrane labeling throughout live and fixed tissue. GLP1R signaling can additionally be evoked when the receptor is allosterically modulated in the presence of LUXendin645. Using LUXendin645 and LUXendin651, we describe islet, brain and hESC-derived β-like cell GLP1R expression patterns, reveal higher-order GLP1R organization including membrane nanodomains, and track single receptor subpopulations. We furthermore show that the LUXendin backbone can be optimized for intravital two-photon imaging by installing a red fluorophore. Thus, our super-resolution compatible labeling probes allow visualization of endogenous GLP1R, and provide insight into class B GPCR distribution and dynamics both in vitro and in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-14309-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6981144PMC
January 2020

Lrrc55 is a novel prosurvival factor in pancreatic islets.

Am J Physiol Endocrinol Metab 2019 11 17;317(5):E794-E804. Epub 2019 Sep 17.

Cumming School of Medicine, Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Canada.

Pancreatic islets adapt to the increase in insulin demand during pregnancy by upregulating β-cell number, insulin synthesis, and secretion. These changes require prolactin receptor (PrlR) signaling, as mice with PrlR deletion are glucose intolerant with a lower β-cell mass. Prolactin also prevents β-cell apoptosis. Many genes participate in these adaptive changes in the islet, and is one of the most upregulated genes with unknown function in islets. Because Lrrc55 expression increases in parallel to the increase in β-cell number and insulin production during pregnancy, we hypothesize that Lrrc55 might regulate β-cell proliferation/apoptosis (thus β-cell number) and insulin synthesis. Here, we found that Lrrc55 expression was upregulated by >60-fold during pregnancy in a PrlR-dependent manner, and this increase was restricted only to the islets. Overexpression of Lrrc55 in β-cells had minimal effect on β-cell proliferation and glucose-stimulated insulin secretion but protected β-cells from glucolipotoxicity-induced reduction in insulin gene expression. Moreover, Lrrc55 protects β-cells from glucolipotoxicity-induced apoptosis, with upregulation of prosurvival signals and downregulation of proapoptotic signals of the endoplasmic reticulum (ER) stress pathway. Furthermore, Lrrc55 attenuated calcium depletion induced by glucolipotoxicity, which may contribute to its antiapoptotic effect. Hence our findings suggest that Lrrc55 is a novel prosurvival factor that is upregulated specifically in islets during pregnancy, and it prevents conversion of adaptive unfolded protein response to unresolved ER stress and apoptosis in β-cells. Lrrc55 could be a potential therapeutic target in diabetes by reducing ER stress and promoting β-cell survival.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajpendo.00028.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879869PMC
November 2019

TrxG Complex Catalytic and Non-catalytic Activity Play Distinct Roles in Pancreas Progenitor Specification and Differentiation.

Cell Rep 2019 08;28(7):1830-1844.e6

Department of Surgery, University of British Columbia, Vancouver, BC V5Z 4E3, Canada; Diabetes Research Group, British Columbia Children's Hospital Research Institute, 950 West 28th Avenue, Vancouver, BC V5Z 4H4, Canada. Electronic address:

Appropriate regulation of genes that coordinate pancreas progenitor proliferation and differentiation is required for pancreas development. Here, we explore the role of H3K4 methylation and the Trithorax group (TrxG) complexes in mediating gene expression during pancreas development. Disruption of TrxG complex assembly, but not catalytic activity, prevented endocrine cell differentiation in pancreas progenitor spheroids. In vivo loss of TrxG catalytic activity in PDX1 cells increased apoptosis and the fraction of progenitors in the G1 phase of the cell cycle. Pancreas progenitors were reallocated to the acinar lineage, primarily at the expense of NEUROG3 endocrine progenitors. Later in development, acinar and endocrine cell numbers were decreased, and increased gene expression variance and reduced terminal marker activation in acinar cells led to their incomplete differentiation. These findings demonstrate that TrxG co-activator activity is required for gene induction, whereas TrxG catalytic activity and H3K4 methylation help maintain transcriptional stability.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2019.07.035DOI Listing
August 2019

In vitro analyses of suspected arrhythmogenic thin filament variants as a cause of sudden cardiac death in infants.

Proc Natl Acad Sci U S A 2019 04 18;116(14):6969-6974. Epub 2019 Mar 18.

Molecular Cardiac Physiology Group, Simon Fraser University, Burnaby, BC V5A 1S6, Canada;

Sudden unexpected death of an infant (SUDI) is a devastating occurrence for families. To investigate the genetic pathogenesis of SUDI, we sequenced >70 genes from 191 autopsy-negative SUDI victims. Ten infants sharing a previously unknown variant in troponin I (TnI) were identified. The mutation ( R37C) is in the fetal/neonatal paralog of TnI, a gene thought to be expressed in the heart up to the first 24 months of life. Using phylogenetic analysis and molecular dynamics simulations, it was determined that arginine at residue 37 in may play a critical functional role, suggesting that the variant may be pathogenic. We investigated the biophysical properties of the R37C mutation in human reconstituted thin filaments (RTFs) using fluorometry. RTFs reconstituted with the mutant R37C TnI exhibited reduced Ca-binding sensitivity due to an increased Ca off-rate constant. Furthermore, we generated R37C mutants in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) using CRISPR-Cas9. In monolayers of hiPSC-CMs, we simultaneously monitored voltage and Ca transients through optical mapping and compared them to their isogenic controls. We observed normal intrinsic beating patterns under control conditions in R37C at stimulation frequencies of 55 beats/min (bpm), but these cells showed no restitution with increased stimulation frequency to 65 bpm and exhibited alternans at >75 bpm. The WT hiPSC-CMs did not exhibit any sign of arrhythmogenicity even at stimulation frequencies of 120 bpm. The approach used in this study provides critical physiological and mechanistic bases to investigate sarcomeric mutations in the pathogenesis of SUDI.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1819023116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6452669PMC
April 2019

Friend and foe: β-cell Ca signaling and the development of diabetes.

Mol Metab 2019 03 24;21:1-12. Epub 2018 Dec 24.

Diabetes Research Group, BC Children's Hospital Research Institute, Vancouver, British Columbia, Canada; Department of Surgery, University of British Columbia, Vancouver, British Columbia, Canada; Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada. Electronic address:

Background: The divalent cation Calcium (Ca) regulates a wide range of processes in disparate cell types. Within insulin-producing β-cells, increases in cytosolic Ca directly stimulate insulin vesicle exocytosis, but also initiate multiple signaling pathways. Mediated through activation of downstream kinases and transcription factors, Ca-regulated signaling pathways leverage substantial influence on a number of critical cellular processes within the β-cell. Additionally, there is evidence that prolonged activation of these same pathways is detrimental to β-cell health and may contribute to Type 2 Diabetes pathogenesis.

Scope Of Review: This review aims to briefly highlight canonical Ca signaling pathways in β-cells and how β-cells regulate the movement of Ca across numerous organelles and microdomains. As a main focus, this review synthesizes experimental data from in vitro and in vivo models on both the beneficial and detrimental effects of Ca signaling pathways for β-cell function and health.

Major Conclusions: Acute increases in intracellular Ca stimulate a number of signaling cascades, resulting in (de-)phosphorylation events and activation of downstream transcription factors. The short-term stimulation of these Ca signaling pathways promotes numerous cellular processes critical to β-cell function, including increased viability, replication, and insulin production and secretion. Conversely, chronic stimulation of Ca signaling pathways increases β-cell ER stress and results in the loss of β-cell differentiation status. Together, decades of study demonstrate that Ca movement is tightly regulated within the β-cell, which is at least partially due to its dual roles as a potent signaling molecule.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molmet.2018.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6407368PMC
March 2019

Single-Cell Transcriptome Profiling of Mouse and hESC-Derived Pancreatic Progenitors.

Stem Cell Reports 2018 12;11(6):1551-1564

Diabetes Research Group, BC Children's Hospital Research Institute, Vancouver, BC V5Z 4H4, Canada; Departments of Surgery and Cellular and Physiological Sciences, University of British Columbia, 950 28(th) Avenue West, Vancouver, BC V5Z4H4, Canada. Electronic address:

Human embryonic stem cells (hESCs) are a potential unlimited source of insulin-producing β cells for diabetes treatment. A greater understanding of how β cells form during embryonic development will improve current hESC differentiation protocols. All pancreatic endocrine cells, including β cells, are derived from Neurog3-expressing endocrine progenitors. This study characterizes the single-cell transcriptomes of 6,905 mouse embryonic day (E) 15.5 and 6,626 E18.5 pancreatic cells isolated from Neurog3-Cre; Rosa26 embryos, allowing for enrichment of endocrine progenitors (yellow; tdTomato + EGFP) and endocrine cells (green; EGFP). Using a NEUROG3-2A-eGFP CyT49 hESC reporter line (N5-5), 4,462 hESC-derived GFP+ cells were sequenced. Differential expression analysis revealed enrichment of markers that are consistent with progenitor, endocrine, or previously undescribed cell-state populations. This study characterizes the single-cell transcriptomes of mouse and hESC-derived endocrine progenitors and serves as a resource (https://lynnlab.shinyapps.io/embryonic_pancreas) for improving the formation of functional β-like cells from hESCs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stemcr.2018.11.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294286PMC
December 2018

Recessive mutations in ATP8A2 cause severe hypotonia, cognitive impairment, hyperkinetic movement disorders and progressive optic atrophy.

Orphanet J Rare Dis 2018 05 31;13(1):86. Epub 2018 May 31.

Division of Clinical and Metabolic Genetics, Department of Pediatrics, The Hospital for Sick Children, University of Toronto, 555 University Avenue, Toronto, ON, M5G 1X8, Canada.

Background: ATP8A2 mutations have recently been described in several patients with severe, early-onset hypotonia and cognitive impairment. The aim of our study was to characterize the clinical phenotype of patients with ATP8A2 mutations.

Methods: An observational study was conducted at multiple diagnostic centres. Clinical data is presented from 9 unreported and 2 previously reported patients with ATP8A2 mutations. We compare their features with 3 additional patients that have been previously reported in the medical literature.

Results: Eleven patients with biallelic ATP8A2 mutations were identified, with a mean age of 9.4 years (range 2.5-28 years). All patients with ATP8A2 mutations (100%) demonstrated developmental delay, severe hypotonia and movement disorders, specifically chorea or choreoathetosis (100%), dystonia (27%) and facial dyskinesia (18%). Optic atrophy was observed in 78% of patients for whom funduscopic examination was performed. Symptom onset in all (100%) was noted before 6 months of age, with 70% having symptoms noted at birth. Feeding difficulties were common (91%) although most patients were able to tolerate pureed or thickened feeds, and 3 patients required gastrostomy tube insertion. MRI of the brain was normal in 50% of the patients. A smaller proportion was noted to have mild cortical atrophy (30%), delayed myelination (20%) and/or hypoplastic optic nerves (20%). Functional studies were performed on differentiated induced pluripotent cells from one child, which confirmed a decrease in ATP8A2 expression compared to control cells.

Conclusions: ATP8A2 gene mutations have emerged as the cause of a novel neurological phenotype characterized by global developmental delays, severe hypotonia and hyperkinetic movement disorders, the latter being an important distinguishing feature. Optic atrophy is common and may only become apparent in the first few years of life, necessitating repeat ophthalmologic evaluation in older children. Early recognition of the cardinal features of this condition will facilitate diagnosis of this complex neurologic disorder.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13023-018-0825-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048855PMC
May 2018

The Polycomb-Dependent Epigenome Controls β Cell Dysfunction, Dedifferentiation, and Diabetes.

Cell Metab 2018 Jun 10;27(6):1294-1308.e7. Epub 2018 May 10.

Max Planck Institute of Immunobiology and Epigenetics, Stuebeweg 51, 79108 Freiburg, Germany. Electronic address:

To date, it remains largely unclear to what extent chromatin machinery contributes to the susceptibility and progression of complex diseases. Here, we combine deep epigenome mapping with single-cell transcriptomics to mine for evidence of chromatin dysregulation in type 2 diabetes. We find two chromatin-state signatures that track β cell dysfunction in mice and humans: ectopic activation of bivalent Polycomb-silenced domains and loss of expression at an epigenomically unique class of lineage-defining genes. β cell-specific Polycomb (Eed/PRC2) loss of function in mice triggers diabetes-mimicking transcriptional signatures and highly penetrant, hyperglycemia-independent dedifferentiation, indicating that PRC2 dysregulation contributes to disease. The work provides novel resources for exploring β cell transcriptional regulation and identifies PRC2 as necessary for long-term maintenance of β cell identity. Importantly, the data suggest a two-hit (chromatin and hyperglycemia) model for loss of β cell identity in diabetes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cmet.2018.04.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5989056PMC
June 2018

Neuronal PAS Domain Protein 4 Suppression of Oxygen Sensing Optimizes Metabolism during Excitation of Neuroendocrine Cells.

Cell Rep 2018 01;22(1):163-174

Diabetes Research Group, BC Children's Hospital Research Institute, Vancouver, BC, Canada; Department of Surgery, University of British Columbia, Vancouver, BC, Canada. Electronic address:

Depolarization of neuroendocrine cells results in calcium influx, which induces vesicle exocytosis and alters gene expression. These processes, along with the restoration of resting membrane potential, are energy intensive. We hypothesized that cellular mechanisms exist to maximize energy production during excitation. Here, we demonstrate that NPAS4, an immediate early basic helix-loop-helix (bHLH)-PAS transcription factor, acts to maximize energy production by suppressing hypoxia-inducible factor 1α (HIF1α). As such, knockout of Npas4 from insulin-producing β cells results in reduced OXPHOS, loss of insulin secretion, β cell dedifferentiation, and type 2 diabetes. NPAS4 plays a similar role in the nutrient-sensing cells of the hypothalamus. Its knockout here results in increased food intake, reduced locomotor activity, and elevated peripheral glucose production. In conclusion, NPAS4 is critical for the coordination of metabolism during the stimulation of electrically excitable cells; its loss leads to the defects in cellular metabolism that underlie the cellular dysfunction that occurs in metabolic disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2017.12.033DOI Listing
January 2018

The p300 and CBP Transcriptional Coactivators Are Required for β-Cell and α-Cell Proliferation.

Diabetes 2018 03 7;67(3):412-422. Epub 2017 Dec 7.

Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada.

p300 () and CBP () are transcriptional coactivators with histone acetyltransferase activity. Various β-cell transcription factors can recruit p300/CBP, and thus the coactivators could be important for β-cell function and health in vivo. We hypothesized that p300/CBP contribute to the development and proper function of pancreatic islets. To test this, we bred and studied mice lacking p300/CBP in their islets. Mice lacking either p300 or CBP in islets developed glucose intolerance attributable to impaired insulin secretion, together with reduced α- and β-cell area and islet insulin content. These phenotypes were exacerbated in mice with only a single copy of p300 or CBP expressed in islets. Removing p300 in pancreatic endocrine progenitors impaired proliferation of neonatal α- and β-cells. Mice lacking all four copies of p300/CBP in pancreatic endocrine progenitors failed to establish α- and β-cell mass postnatally. Transcriptomic analyses revealed significant overlaps between p300/CBP-downregulated genes and genes downregulated in Hnf1α-null islets and Nkx2.2-null islets, among others. Furthermore, p300/CBP are important for the acetylation of H3K27 at loci downregulated in Hnf1α-null islets. We conclude that p300 and CBP are limiting cofactors for islet development, and hence for postnatal glucose homeostasis, with some functional redundancy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2337/db17-0237DOI Listing
March 2018

SOX4 Allows Facultative β-Cell Proliferation Through Repression of .

Diabetes 2017 08 11;66(8):2213-2219. Epub 2017 May 11.

BC Children's Hospital Research Institute, Vancouver, British Columbia, Canada

The high-mobility group box transcription factor SOX4 is the most highly expressed SOX family protein in pancreatic islets, and mutations in are associated with an increased risk of developing type 2 diabetes. We used an inducible β-cell knockout mouse model to test the hypothesis that is essential for the maintenance of β-cell number during the development of type 2 diabetes. Knockout of at 6 weeks of age resulted in time-dependent worsening of glucose tolerance, impairment of insulin secretion, and diabetes by 30 weeks of age. Immunostaining revealed a decrease in β-cell mass in knockout mice that was caused by a 39% reduction in β-cell proliferation. Gene expression studies revealed that induction of the cell cycle inhibitor was responsible for the decreased proliferation in the knockout animals. Altogether, this study demonstrates that SOX4 is necessary for adult β-cell replication through direct regulation of the β-cell cycle.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2337/db16-1074DOI Listing
August 2017

Phosphorylation of NEUROG3 Links Endocrine Differentiation to the Cell Cycle in Pancreatic Progenitors.

Dev Cell 2017 04;41(2):129-142.e6

Diabetes Research Group, BC Children's Hospital Research Institute, Vancouver, BC V5Z 4H4, Canada; Departments of Surgery and Cellular and Physiological Sciences, University of British Columbia, 950 28th Avenue West, Vancouver, BC V5Z 4H4, Canada. Electronic address:

During pancreatic development, proliferating pancreatic progenitors activate the proendocrine transcription factor neurogenin 3 (NEUROG3), exit the cell cycle, and differentiate into islet cells. The mechanisms that direct robust NEUROG3 expression within a subset of progenitor cells control the size of the endocrine population. Here we demonstrate that NEUROG3 is phosphorylated within the nucleus on serine 183, which catalyzes its hyperphosphorylation and proteosomal degradation. During progression through the progenitor cell cycle, NEUROG3 phosphorylation is driven by the actions of cyclin-dependent kinases 2 and 4/6 at G/S cell-cycle checkpoint. Using models of mouse and human pancreas development, we show that lengthening of the G phase of the pancreatic progenitor cell cycle is essential for proper induction of NEUROG3 and initiation of endocrine cell differentiation. In sum, these studies demonstrate that progenitor cell-cycle G lengthening, through its actions on stabilization of NEUROG3, is an essential variable in normal endocrine cell genesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.devcel.2017.02.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5517315PMC
April 2017

Generation of a Conditional Allele of the Transcription Factor Atonal Homolog 8 (Atoh8).

PLoS One 2016 11;11(1):e0146273. Epub 2016 Jan 11.

Diabetes and Obesity Research Laboratory, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain.

Atonal Homolog 8 (Atoh8) is a basic helix-loop-helix (bHLH) transcription factor that is highly conserved across species and expressed in multiple tissues during embryogenesis. In the developing pancreas, Atoh8 is expressed in endocrine progenitors but declines in hormone-positive cells, suggesting a role during early stages of the endocrine differentiation program. We previously generated a whole-body Atoh8 knockout but early lethality of null embryos precluded assessment of Atoh8 functions during organ development. Here we report the generation of a conditional Atoh8 knockout mouse strain by insertion of two loxP sites flanking exon 1 of the Atoh8 gene. Pancreas-specific Atoh8 knockout (Atoh8 Δpanc) mice were obtained by mating this strain with a Pdx1-Cre transgenic line. Atoh8 Δpanc mice were born at the expected mendelian ratio and showed normal appearance and fertility. Pancreas weight and gross pancreatic morphology were normal. All pancreatic cell lineages were present, although endocrine δ (somatostatin) cells were modestly augmented in Atoh8 Δpanc as compared to control neonates. This increase did not affect whole-body glucose tolerance in adult knockout animals. Gene expression analysis in embryonic pancreases at the time of the major endocrine differentiation wave revealed modest alterations in several early endocrine differentiation markers. Together, these data argue that Atoh8 modulates activation of the endocrine program but it is not essential for pancreas formation or endocrine differentiation in the mouse. Given the ubiquitous expression pattern of Atoh8, the availability of a mouse strain carrying a conditional allele for this gene warrants further studies using temporally regulated Cre transgenic lines to elucidate time or cell-autonomous functions of Atoh8 during development and in the adult.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0146273PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4708992PMC
July 2016

Reawakening the Duct Cell Progenitor?

Endocrinology 2016 Jan;157(1):52-3

Departments of Surgery (C.B.V., F.C.L.) and Pathology and Laboratory Medicine (C.B.V.), Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada V5Z4H4.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/en.2015-2008DOI Listing
January 2016

Npas4 Transcription Factor Expression Is Regulated by Calcium Signaling Pathways and Prevents Tacrolimus-induced Cytotoxicity in Pancreatic Beta Cells.

J Biol Chem 2016 Feb 9;291(6):2682-95. Epub 2015 Dec 9.

From the Diabetes Research Program, Child and Family Research Institute, Vancouver, British Columbia V5Z 4H4, Canada and the Department of Surgery and Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia V5Z 1M9, Canada

Cytosolic calcium influx activates signaling pathways known to support pancreatic beta cell function and survival by modulating gene expression. Impaired calcium signaling leads to decreased beta cell mass and diabetes. To appreciate the causes of these cytotoxic perturbations, a more detailed understanding of the relevant signaling pathways and their respective gene targets is required. In this study, we examined the calcium-induced expression of the cytoprotective beta cell transcription factor Npas4. Pharmacological inhibition implicated the calcineurin, Akt/protein kinase B, and Ca(2+)/calmodulin-dependent protein kinase signaling pathways in the regulation of Npas4 transcription and translation. Both Npas4 mRNA and protein had high turnover rates, and, at the protein level, degradation was mediated via the ubiquitin-proteasome pathway. Finally, beta cell cytotoxicity of the calcineurin inhibitor and immunosuppressant tacrolimus (FK-506) was prevented by Npas4 overexpression. These results delineate the pathways regulating Npas4 expression and stability and demonstrate its importance in clinical settings such as islet transplantation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M115.704098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4742737PMC
February 2016

Reduced Insulin Production Relieves Endoplasmic Reticulum Stress and Induces β Cell Proliferation.

Cell Metab 2016 Jan 25;23(1):179-93. Epub 2015 Nov 25.

Department of Cellular and Physiological Sciences, Diabetes Research Group, Life Sciences Institute, University of British Columbia, BC V6T1Z3, Canada. Electronic address:

Pancreatic β cells are mostly post-mitotic, but it is unclear what locks them in this state. Perturbations including uncontrolled hyperglycemia can drive β cells into more pliable states with reduced cellular insulin levels, increased β cell proliferation, and hormone mis-expression, but it is unknown whether reduced insulin production itself plays a role. Here, we define the effects of ∼50% reduced insulin production in Ins1(-/-):Ins2(f/f):Pdx1Cre(ERT):mTmG mice prior to robust hyperglycemia. Transcriptome, proteome, and network analysis revealed alleviation of chronic endoplasmic reticulum (ER) stress, indicated by reduced Ddit3, Trib3, and Atf4 expression; reduced Xbp1 splicing; and reduced phospho-eIF2α. This state was associated with hyper-phosphorylation of Akt, which is negatively regulated by Trib3, and with cyclinD1 upregulation. Remarkably, β cell proliferation was increased 2-fold after reduced insulin production independently of hyperglycemia. Eventually, recombined cells mis-expressed glucagon in the hyperglycemic state. We conclude that the normally high rate of insulin production suppresses β cell proliferation in a cell-autonomous manner.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cmet.2015.10.016DOI Listing
January 2016

Use-dependent activation of neuronal Kv1.2 channel complexes.

J Neurosci 2015 Feb;35(8):3515-24

Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, Canada, V6T 1Z3

In excitable cells, ion channels are frequently challenged by repetitive stimuli, and their responses shape cellular behavior by regulating the duration and termination of bursts of action potentials. We have investigated the behavior of Shaker family voltage-gated potassium (Kv) channels subjected to repetitive stimuli, with a particular focus on Kv1.2. Genetic deletion of this subunit results in complete mortality within 2 weeks of birth in mice, highlighting a critical physiological role for Kv1.2. Kv1.2 channels exhibit a unique property described previously as "prepulse potentiation," in which activation by a depolarizing step facilitates activation in a subsequent pulse. In this study, we demonstrate that this property enables Kv1.2 channels to exhibit use-dependent activation during trains of very brief depolarizations. Also, Kv subunits usually assemble into heteromeric channels in the central nervous system, generating diversity of function and sensitivity to signaling mechanisms. We demonstrate that other Kv1 channel types do not exhibit use-dependent activation, but this property is conferred in heteromeric channel complexes containing even a single Kv1.2 subunit. This regulatory mechanism is observed in mammalian cell lines as well as primary cultures of hippocampal neurons. Our findings illustrate that use-dependent activation is a unique property of Kv1.2 that persists in heteromeric channel complexes and may influence function of hippocampal neurons.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.4518-13.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6605548PMC
February 2015

SOX4 cooperates with neurogenin 3 to regulate endocrine pancreas formation in mouse models.

Diabetologia 2015 May 5;58(5):1013-23. Epub 2015 Feb 5.

Diabetes Research Program, Child and Family Research Institute, A4-184, 950 West 28 Ave, Vancouver, BC, V5Z 4H4, Canada.

Aims/hypothesis: The sex-determining region Y (SRY)-related high mobility group (HMG) box (SOX) family of transcription factors is essential for normal organismal development. Despite the longstanding knowledge that many SOX family members are expressed during pancreas development, a role for many of these factors in the establishment of insulin-producing beta cell fate remains to be determined. The aim of this study is to elucidate the role of SOX4 during beta cell development.

Methods: We used pancreas and endocrine progenitor mouse knockouts of Sox4 to uncover the roles of SOX4 during pancreas development. Lineage tracing and in vitro models were used to determine how SOX4 regulates beta cell formation and understand the fate of Sox4-null endocrine lineage cells.

Results: This study demonstrates a progenitor cell-autonomous role for SOX4 in regulating the genesis of beta cells and shows that it is required at multiple stages of the process. SOX4 deletion in the multipotent pancreatic progenitors resulted in impaired endocrine progenitor cell differentiation. Deletion of SOX4 later in the Neurog3-expressing cells also caused reductions in beta cells. Lineage studies showed loss of Sox4 in endocrine progenitors resulted in a block in terminal islet cell differentiation that was attributed to reduction in the production of key beta cell specification factors.

Conclusions/interpretation: These results demonstrate that SOX4 is essential for normal endocrine pancreas development both concomitant with, and downstream of, the endocrine fate decision. In conclusion, these studies position Sox4 temporally in the endocrine differentiation programme and provide a new target for improving in vitro differentiation of glucose-responsive pancreatic beta cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00125-015-3507-xDOI Listing
May 2015

All-encomPASsing regulation of β-cells: PAS domain proteins in β-cell dysfunction and diabetes.

Trends Endocrinol Metab 2015 Jan 11;26(1):49-57. Epub 2014 Dec 11.

Diabetes Research Group, Child and Family Research Institute, Vancouver, British Columbia, Canada; The Departments of Surgery and Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, V5Z 4H4 Canada. Electronic address:

As a sensory micro-organ, pancreatic β-cells continually respond to nutritional signals and neuroendocrine input from other glucoregulatory organs. This sensory ability is essential for normal β-cell function and systemic glucose homeostasis. Period circadian protein (Per)-aryl hydrocarbon receptor nuclear translocator protein (Arnt)-single-minded protein (Sim) (PAS) domain proteins have a conserved role as sensory proteins, critical in adaptation to changes in voltage, oxygen potential, and xenobiotics. Within β-cells, PAS domain proteins such as hypoxia inducible factor 1α (Hif1α), Arnt, PAS kinase, Bmal1, and Clock respond to disparate stimuli, but act in concert to maintain proper β-cell function. Elucidating the function of these factors in islets offers a unique insight into the sensing capacity of β-cells, the consequences of impaired sensory function, and the potential to develop novel therapeutic targets for preserving β-cell function in diabetes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.tem.2014.11.002DOI Listing
January 2015

TALEN/CRISPR-mediated eGFP knock-in add-on at the OCT4 locus does not impact differentiation of human embryonic stem cells towards endoderm.

PLoS One 2014 4;9(12):e114275. Epub 2014 Dec 4.

Diabetes Research Program, Child and Family Research Institute, Vancouver, British Columbia, Canada; Department of Surgery and Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada.

Human embryonic stem cells (hESCs) have great promise as a source of unlimited transplantable cells for regenerative medicine. However, current progress on producing the desired cell type for disease treatment has been limited due to an insufficient understanding of the developmental processes that govern their differentiation, as well as a paucity of tools to systematically study differentiation in the lab. In order to overcome these limitations, cell-type reporter hESC lines will be required. Here we outline two strategies using Transcription Activator Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-Associated protein (Cas) to create OCT4-eGFP knock-in add-on hESC lines. Thirty-one and forty-seven percent of clones were correctly modified using the TALEN and CRISPR-Cas9 systems, respectively. Further analysis of three correctly targeted clones demonstrated that the insertion of eGFP in-frame with OCT4 neither significantly impacted expression from the wild type allele nor did the fusion protein have a dramatically different biological stability. Importantly, the OCT4-eGFP fusion was easily detected using microscopy, flow cytometry and western blotting. The OCT4 reporter lines remained equally competent at producing CXCR4+ definitive endoderm that expressed a panel of endodermal genes. Moreover, the genomic modification did not impact the formation of NKX6.1+/SOX9+ pancreatic progenitor cells following directed differentiation. In conclusion, these findings demonstrate for the first time that CRISPR-Cas9 can be used to modify OCT4 and highlight the feasibility of creating cell-type specific reporter hESC lines utilizing genome-editing tools that facilitate homologous recombination.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0114275PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4256397PMC
December 2015

Glycoprotein 130 receptor signaling mediates α-cell dysfunction in a rodent model of type 2 diabetes.

Diabetes 2014 Sep 8;63(9):2984-95. Epub 2014 May 8.

Department of Surgery, Faculty of Medicine, University of British Columbia, Child and Family Research Institute, Vancouver, British Columbia, Canada

Dysregulated glucagon secretion accompanies islet inflammation in type 2 diabetes. We recently discovered that interleukin (IL)-6 stimulates glucagon secretion from human and rodent islets. IL-6 family cytokines require the glycoprotein 130 (gp130) receptor to signal. In this study, we elucidated the effects of α-cell gp130 receptor signaling on glycemic control in type 2 diabetes. IL-6 family cytokines were elevated in islets in rodent models of this disease. gp130 receptor activation increased STAT3 phosphorylation in primary α-cells and stimulated glucagon secretion. Pancreatic α-cell gp130 knockout (αgp130KO) mice showed no differences in glycemic control, α-cell function, or α-cell mass. However, when subjected to streptozotocin plus high-fat diet to induce islet inflammation and pathophysiology modeling type 2 diabetes, αgp130KO mice had reduced fasting glycemia, improved glucose tolerance, reduced fasting insulin, and improved α-cell function. Hyperinsulinemic-euglycemic clamps revealed no differences in insulin sensitivity. We conclude that in a setting of islet inflammation and pathophysiology modeling type 2 diabetes, activation of α-cell gp130 receptor signaling has deleterious effects on α-cell function, promoting hyperglycemia. Antagonism of α-cell gp130 receptor signaling may be useful for the treatment of type 2 diabetes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2337/db13-1121DOI Listing
September 2014

Quetiapine treatment in youth is associated with decreased insulin secretion.

J Clin Psychopharmacol 2014 Jun;34(3):359-64

From the *Department of Pediatrics, Faculty of Medicine, University of British Columbia; †Child & Family Research Institute; Departments of ‡Surgery, and §Psychiatry, Faculty of Medicine, University of British Columbia, Vancouver, Canada.

Second-generation antipsychotics (SGAs) are commonly prescribed to youth but are associated with metabolic effects including obesity and diabetes. The mechanisms underlying diabetes development are unclear. The purpose of this study was to compare glucose homeostasis, insulin sensitivity, insulin secretion, and overall β-cell function in risperidone-treated, quetiapine-treated, and SGA-naive youth with mental illness. We conducted a cross-sectional study in which youth aged 9 to 18 years underwent a 2-hour oral glucose tolerance test. Indices for insulin sensitivity (Matsuda index), insulin secretion (insulinogenic index), and β-cell function (insulin secretion-sensitivity index-2 [ISSI-2]) were calculated. A total of 18 SGA-naive, 20 risperidone-treated, and 16 quetiapine-treated youth participated. The 3 groups were similar in age, sex, ethnicity, body mass index standardized for age and sex, pubertal status, degree of psychiatric illness, psychiatric diagnoses, and other medications. The median treatment duration was 17 months (range, 3-91 months) for risperidone-treated youth and 10 months (range, 3-44 months) for quetiapine-treated youth. The quetiapine-treated group had lower insulinogenic index (P < 0.01) and lower ISSI-2 (P < 0.01) compared with that in the SGA-naive group. Only the body mass index standardized for age and sex was negatively associated with Matsuda index (β = -0.540, P < 0.001) in all youth. Quetiapine treatment was negatively associated with insulinogenic index (β = -0.426, P = 0.007) and ISSI-2 (β = -0.433, P = 0.008). Quetiapine reduced the insulin expression in isolated mouse islets suggesting a direct β-cell effect. Our results suggest that quetiapine treatment in youth is associated with impaired β-cell function, specifically lower insulin secretion. Prospective longitudinal studies are required to understand the progression of β-cell dysfunction after quetiapine initiation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/JCP.0000000000000118DOI Listing
June 2014

Characterization of polyhormonal insulin-producing cells derived in vitro from human embryonic stem cells.

Stem Cell Res 2014 Jan 16;12(1):194-208. Epub 2013 Oct 16.

Laboratory of Molecular and Cellular Medicine, Department of Cellular & Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada; Department of Surgery, University of British Columbia, Vancouver, BC, Canada. Electronic address:

Human embryonic stem cells (hESCs) were used as a model system of human pancreas development to study characteristics of the polyhormonal cells that arise during fetal pancreas development. HESCs were differentiated into fetal-like pancreatic cells in vitro using a 33-day, 7-stage protocol. Cultures were ~90-95% PDX1-positive by day (d) 11 and 70-75% NKX6.1-positive by d17. Polyhormonal cells were scattered at d17, but developed into islet-like clusters that expressed key transcription factors by d33. Human C-peptide and glucagon secretion were first detected at d17 and increased thereafter in parallel with INS and GCG transcript levels. HESC-derived cells were responsive to KCl and arginine, but not glucose in perifusion studies. Compared to adult human islets, hESC-derived cells expressed ~10-fold higher levels of glucose transporter 1 (GLUT1) mRNA, but similar levels of glucokinase (GCK). In situ hybridization confirmed the presence of GLUT1 transcript within endocrine cells. However, GLUT1 protein was excluded from this population and was instead observed predominantly in non-endocrine cells, whereas GCK was co-expressed in insulin-positive cells. In rubidium efflux assays, hESC-derived cells displayed mild potassium channel activity, but no responsiveness to glucose, metabolic inhibitors or glibenclamide. Western blotting experiments revealed that the higher molecular weight SUR1 band was absent in hESC-derived cells, suggesting a lack of functional KATP channels at the cell surface. In addition, KATP channel subunit transcript levels were not at a 1:1 ratio, as would be expected (SUR1 levels were ~5-fold lower than KIR6.2). Various ratios of SUR1:KIR6.2 plasmids were transfected into COSM6 cells and rubidium efflux was found to be particularly sensitive to a reduction in SUR1. These data suggest that an impaired ratio of SUR1:KIR6.2 may contribute to the observed KATP channel defects in hESC-derived islet endocrine cells, and along with lack of GLUT1, may explain the absence of glucose-stimulated insulin secretion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.scr.2013.10.003DOI Listing
January 2014

The transcription factor Atonal homolog 8 regulates Gata4 and Friend of Gata-2 during vertebrate development.

J Biol Chem 2013 Aug 8;288(34):24429-40. Epub 2013 Jul 8.

Department of Medicine and Cardiovascular Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

GATA and Friend of GATA (FOG) form a transcriptional complex that plays a key role in cardiovascular development in both fish and mammals. In the present study we demonstrate that the basic helix-loop-helix transcription factor Atonal homolog 8 (Atoh8) is required for development of the heart in fish but not in mice. Genetic studies reveal that Atoh8 interacts specifically with Gata4 and Fog1 during development of the heart and swim bladder in the fish. Biochemical studies reveal that ATOH8, GATA4, and FOG2 associate in a single complex in vitro. In contrast to fish, ATOH8-deficient mice exhibit normal cardiac development and loss of ATOH8 does not alter cardiac development in Gata4(+/-) mice. This species difference in the role of ATOH8 is explained in part by LacZ and GFP reporter alleles that reveal restriction of Atoh8 expression to atrial but not ventricular myocardium in the mouse. Our findings identify ATOH8 as a novel regulator of GATA-FOG function that is required for cardiac development in the fish but not the mouse. Whether ATOH8 modulates GATA-FOG function at other sites or in more subtle ways in mammals is not yet known.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M113.463083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3750143PMC
August 2013

Npas4 is a novel activity-regulated cytoprotective factor in pancreatic β-cells.

Diabetes 2013 Aug 8;62(8):2808-20. Epub 2013 May 8.

Diabetes Research Group, Child and Family Research Institute, Vancouver, British Columbia, Canada.

Cellular homeostasis requires intrinsic sensing mechanisms to temper function in the face of prolonged activity. In the pancreatic β-cell, glucose is likely a physiological trigger that activates an adaptive response to stimulation, thereby maintaining cellular homeostasis. Immediate early genes (IEGs) are activated as a first line of defense in cellular homeostasis and are largely responsible for transmitting an environmental cue to a cellular response. Here we examine the regulation and function of the novel β-cell IEG, neuronal PAS domain protein 4 (Npas4). Using MIN6 cells, mouse and human islets, as well as in vivo infusions, we demonstrate that Npas4 is expressed within pancreatic islets and is upregulated by β-cell depolarizing agents. Npas4 tempers β-cell function through a direct inhibitory interaction with the insulin promoter and by blocking the potentiating effects of GLP-1 without significantly reducing glucose-stimulated secretion. Finally, Npas4 expression is induced by classical endoplasmic reticulum (ER) stressors and can prevent thapsigargin- and palmitate-induced dysfunction and cell death. These results suggest that Npas4 is a key activity-dependent regulator that improves β-cell efficiency in the face of stress. We posit that Npas4 could be a novel therapeutic target in type 2 diabetes that could both reduce ER stress and cell death and maintain basal cell function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2337/db12-1527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3717850PMC
August 2013

Maintenance of β-cell maturity and plasticity in the adult pancreas: developmental biology concepts in adult physiology.

Diabetes 2012 Jun;61(6):1365-71

Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2337/db11-1361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3357305PMC
June 2012

Regulation of GIP and GLP1 receptor cell surface expression by N-glycosylation and receptor heteromerization.

PLoS One 2012 7;7(3):e32675. Epub 2012 Mar 7.

Cardiovascular Research Group, University of British Columbia, Vancouver, British Columbia, Canada.

In response to a meal, Glucose-dependent Insulinotropic Polypeptide (GIP) and Glucagon-like Peptide-1 (GLP-1) are released from gut endocrine cells into the circulation and interact with their cognate G-protein coupled receptors (GPCRs). Receptor activation results in tissue-selective pleiotropic responses that include augmentation of glucose-induced insulin secretion from pancreatic beta cells. N-glycosylation and receptor oligomerization are co-translational processes that are thought to regulate the exit of functional GPCRs from the ER and their maintenance at the plasma membrane. Despite the importance of these regulatory processes, their impact on functional expression of GIP and GLP-1 receptors has not been well studied. Like many family B GPCRs, both the GIP and GLP-1 receptors possess a large extracellular N-terminus with multiple consensus sites for Asn-linked (N)-glycosylation. Here, we show that each of these Asn residues is glycosylated when either human receptor is expressed in Chinese hamster ovary cells. N-glycosylation enhances cell surface expression and function in parallel but exerts stronger control over the GIP receptor than the GLP-1 receptor. N-glycosylation mainly lengthens receptor half-life by reducing degradation in the endoplasmic reticulum. N-glycosylation is also required for expression of the GIP receptor at the plasma membrane and efficient GIP potentiation of glucose-induced insulin secretion from the INS-1 pancreatic beta cell line. Functional expression of a GIP receptor mutant lacking N-glycosylation is rescued by co-expressed wild type GLP1 receptor, which, together with data obtained using Bioluminescence Resonance Energy Transfer, suggests formation of a GIP-GLP1 receptor heteromer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0032675PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3296735PMC
August 2012
-->