scholarly journals Inability to process and store proinsulin in transdifferentiated pancreatic acinar cells lacking the regulated secretory pathway

2007 ◽  
Vol 196 (1) ◽  
pp. 33-43 ◽  
Author(s):  
A Alidibbiat ◽  
C E Marriott ◽  
K T Scougall ◽  
S C Campbell ◽  
G C Huang ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Immunocytochemical Staining ◽  
Cell Phenotype ◽  
Wild Type ◽  
Rt Pcr ◽  
Β Cells ◽  
Β Cell ◽  
Over Expression ◽  
Ar42j Cells ◽  
Regulated Secretory Pathway

Generation of new β-cells from the adult pancreas or the embryonic stem cells is being pursued by research groups worldwide. Success will be dependent on confirmation of true β-cell phenotype evidenced by capacity to process and store proinsulin. The aim of these studies was to robustly determine endocrine characteristics of the AR42J rat pancreatic acinar cell line before and after in vitro transdifferentiation. β-cell phenotypic marker expression was characterised by RT-PCR, immunostaining, western blotting, ELISA and in human preproinsulin transgene over-expression studies in wild-type AR42J cells and after culture on Matrigel basement membrane matrix with and without growth/differentiation factor supplementation. Pancreatic duodenal homeobox 1 (PDX1), forkhead box transcription factor a2 (Foxa2), glucokinase, pancreatic polypeptide and low-level insulin gene transcription in wild-type AR42J cells were confirmed by RT-PCR. Culture on Matrigel-coated plates and supplementation of medium with glucagon-like peptide 1 induced expression of the β-cell Glut 2 with maintained expression of insulin and PDX1. Increased biosynthesis and secretion of proinsulin were confirmed by immunocytochemical staining and sensitive ELISA. Absence of the regulated secretory pathway was demonstrated by undetectable prohormone convertase expression. In addition, inability to process and store endogenous proinsulin or human proinsulin translated from a constitutively over-expressed preproinsulin transgene was confirmed. The importance of robust phenotypic characterisation at the protein level in attempted β-cell transdifferentiation studies has been confirmed. Rodent and human sensitive/specific differential proinsulin/insulin ELISA in combination with human preproinsulin over-expression enables detailed elucidatation of core endocrine functions of proinsulin processing and storage in putative new β-cells.

scholarly journals Mutant Proinsulin That Cannot Be Converted Is Secreted Efficiently from Primary Rat β-Cells via the Regulated Pathway

2003 ◽  
Vol 14 (3) ◽  
pp. 1195-1203 ◽  
Author(s):  
Philippe A. Halban ◽  
Jean-Claude Irminger
Keyword(s):  
High Performance ◽  
Secretory Pathway ◽  
Recombinant Adenovirus ◽  
Islet Cells ◽  
Secretory Granules ◽  
Wild Type ◽  
Β Cells ◽  
Human Proinsulin ◽  
Regulated Secretory Pathway ◽  
Golgi Network

Prohormones are directed from the trans-Golgi network to secretory granules of the regulated secretory pathway. It has further been proposed that prohormone conversion by endoproteolysis may be necessary for subsequent retention of peptides in granules and to prevent their release by the so-called “constitutive-like” pathway. To address this directly, mutant human proinsulin (Arg/Gly32:Lys/Thr64), which cannot be cleaved by conversion endoproteases, was expressed in primary rat islet cells by recombinant adenovirus. The handling of the mutant proinsulin was compared with that of wild-type human proinsulin. Infected islet cells were pulse labeled and both basal and stimulated secretion of radiolabeled products followed during a chase. Labeled products were quantified by high-performance liquid chromatography. As expected, the mutant proinsulin was not converted at any time. Basal (constitutive and constitutive-like) secretion was higher for the mutant proinsulin than for wild-type proinsulin/insulin, but amounted to <1% even during a prolonged (6-h) period of basal chase. There was no difference in stimulated (regulated) secretion of mutant and wild-type proinsulin/insulin at any time. Thus, in primary islet cells, unprocessed (mutant) proinsulin is sorted to the regulated pathway and then retained in secretory granules as efficiently as fully processed insulin.

scholarly journals Furin controls β cell function via mTORC1 signaling

2020 ◽  
Author(s):  
Bas Brouwers ◽  
Ilaria Coppola ◽  
Katlijn Vints ◽  
Bastian Dislich ◽  
Nathalie Jouvet ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Cell Function ◽  
Mammalian Target Of Rapamycin ◽  
Human Islets ◽  
Β Cells ◽  
Β Cell ◽  
Differential Proteomics ◽  
Atpase Subunit ◽  
Proteolytic Activation ◽  
Mtorc1 Signaling

AbstractFurin is a proprotein convertase (PC) responsible for proteolytic activation of a wide array of precursor proteins within the secretory pathway. It maps to the PRC1 locus, a type 2 diabetes susceptibility locus, yet its specific role in pancreatic β cells is largely unknown. The aim of this study was to determine the role of furin in glucose homeostasis. We show that furin is highly expressed in human islets, while PCs that potentially could provide redundancy are expressed at considerably lower levels. β cell-specific furin knockout (βfurKO) mice are glucose intolerant, due to smaller islets with lower insulin content and abnormal dense core secretory granule morphology. RNA expression analysis and differential proteomics on βfurKO islets revealed activation of Activating Transcription Factor 4 (ATF4), which was mediated by mammalian target of rapamycin C1 (mTORC1). βfurKO cells show impaired cleavage of the essential V-ATPase subunit Ac45, and by blocking this pump in β cells the mTORC1 pathway is activated. Furthermore, βfurKO cells show lack of insulin receptor cleavage and impaired response to insulin. Taken together, these results suggest a model of mTORC1-ATF4 hyperactivation in β cells lacking furin, which causes β cell dysfunction.

scholarly journals Role of Nitric Oxide in the Pathogenesis of Encephalomyocarditis Virus-Induced Diabetes in Mice

2009 ◽  
Vol 83 (16) ◽  
pp. 8004-8011 ◽  
Author(s):  
Young-Sun Lee ◽  
Na Li ◽  
Seungjin Shin ◽  
Hee-Sook Jun
Keyword(s):  
Virus Infection ◽  
Encephalomyocarditis Virus ◽  
Wild Type ◽  
Β Cells ◽  
Β Cell ◽  
Pancreatic Β Cells ◽  
Diabetes In Mice ◽  
Tnf Α ◽  
Deficient Mice

ABSTRACT The D variant of encephalomyocarditis virus (EMC-D virus) causes diabetes in mice by destroying pancreatic β cells. In mice infected with a low dose of EMC-D virus, macrophages play an important role in β-cell destruction by producing soluble mediators such as interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), and nitric oxide (NO). To investigate the role of NO and inducible NO synthase (iNOS) in the development of diabetes in EMC-D virus-infected mice, we infected iNOS-deficient DBA/2 mice with EMC-D virus (2 × 102 PFU/mouse). Mean blood glucose levels in EMC-D virus-infected iNOS-deficient mice and wild-type mice were 205.5 and 466.7 mg/dl, respectively. Insulitis and macrophage infiltration were reduced in islets of iNOS-deficient mice compared with wild-type mice at 3 days after EMC-D virus infection. Apoptosis of β cells was decreased in iNOS-deficient mice, as evidenced by reduced numbers of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells. There were no differences in mRNA expression of antiapoptotic molecules Bcl-2, Bcl-xL, Bcl-w, Mcl-1, cIAP-1, and cIAP-2 between wild-type and iNOS-deficient mice, whereas expression of proapoptotic Bax and Bak mRNAs was significantly decreased in iNOS-deficient mice. Expression of IL-1β and TNF-α mRNAs was significantly decreased in both islets and macrophages of iNOS-deficient mice compared with wild-type mice after EMC-D virus infection. Nuclear factor κB was less activated in macrophages of iNOS-deficient mice after virus infection. We conclude that NO plays an important role in the activation of macrophages and apoptosis of pancreatic β cells in EMC-D virus-infected mice and that deficient iNOS gene expression inhibits macrophage activation and β-cell apoptosis, contributing to prevention of EMC-D virus-induced diabetes.

SerpinB1 improves insulin sensitivity

2016 ◽  
Vol 9 (411) ◽  
pp. ec10-ec10
Author(s):  
Annalisa M. VanHook
Keyword(s):  
Cell Proliferation ◽  
Insulin Sensitivity ◽  
Cell Mass ◽  
Dependent Manner ◽  
Wild Type ◽  
Β Cells ◽  
Β Cell ◽  
Cell Hyperplasia ◽  
Link Type ◽  
Proteomic Approach

Pancreatic β cells adjust the secretion of insulin in response to acute changes in plasma glucose concentration. These cells also compensate for long-term changes in insulin sensitivity by adjusting their activity or numbers, or both (see Tarasov and Rorsman). In addition to being insulin resistant, mice lacking the liver insulin receptor (LIRKO mice) also exhibit β cell hyperplasia that depends on factors released from the liver. Using a proteomic approach, El Ouaamari etal. found that the abundance of the protease inhibitor serpinB1 was greater in liver extracts, liver explant–conditioned medium, and serum from LIRKO mice than in those from wild-type mice. SerpinB1 abundance correlated inversely with insulin sensitivity in human patients with risk factors for type 2 diabetes. Recombinant human serpinB1 stimulated the proliferation of β cells in cultured mouse and human islets in a dose-dependent manner. Elastase is a protease inhibited by serpinB1, and forms of serpinB1 that do not inhibit elastase activity did not stimulate proliferation of cultured mouse β cells. Compounds that inhibit elastase also promoted the proliferation of cultured mouse β cells. In mice, elastase inhibitors stimulated the proliferation of both endogenous β cells and the β cells of human islet grafts. Furthermore, overexpression of serpinb1 increased the regeneration of β cells following β cell ablation in zebrafish embryos. In several models of acute and chronic insulin resistance, serpinb1 knockout mice exhibited reduced β cell proliferation compared with wild-type controls. However, β cell proliferation was not abolished in serpinb1 knockouts, indicating that additional factors can induce compensatory proliferation of β cells. Phosphoproteomic analyses demonstrated that treatment of cultured mouse β cells with human serpinB1 stimulated signaling through several pathways that promote cell proliferation and survival. Commentary by Tarasov and Rorsman considers how these findings might be put to clinical use.A. El Ouaamari, E. Dirice, N. Gedeon, J. Hu, J.-Y. Zhou, J. Shirakawa, L. Hou, J. Goodman, C. Karampelias, G. Qiang, J. Boucher, R. Martinez, M. A. Gritsenko, D. F. De Jesus, S. Kahraman, S. Bhatt, R. D. Smith, H.-D. Beer, P. Jungtrakoon, Y. Gong, A. B. Goldfine, C. W. Liew, A. Doria, O. Andersson, W.-J. Qian, E. Remold-O’Donnell, R. N. Kulkarni, SerpinB1 promotes pancreatic β cell proliferation. CellMetab. 23, 194–205 (2016). [PubMed] A. I. Tarasov, P. Rorsman, Dramatis personae in β-cell mass regulation: Enter SerpinB1. CellMetab. 23, 8–10 (2016). [Online Journal]

scholarly journals The pro region is not required for the expression or intracellular routeing of carboxypeptidase E

1997 ◽  
Vol 323 (1) ◽  
pp. 265-271 ◽  
Author(s):  
Lixin SONG ◽  
Lloyd D. FRICKER
Keyword(s):  
Confocal Microscopy ◽  
Secretory Pathway ◽  
Secretory Vesicles ◽  
Wild Type ◽  
Carboxypeptidase E ◽  
N Terminus ◽  
Regulated Secretory Pathway ◽  
Golgi Network ◽  
Pro Region ◽  
Stimulation Of

Carboxypeptidase E (CPE) is initially synthesized as a larger precursor containing an additional 14-residue propeptide that is highly conserved between human and rat. Previous studies have established that the proenzyme is enzymically active and that deletion of the pro region does not affect the expression of the active enzyme. In the present study the function of the pro region was examined both by deleting this region from CPE and by attaching this region to the N-terminus of albumin. CPE lacking the pro region is sorted into the regulated secretory pathway in AtT-20 cells, based on confocal microscopy and examination of the stimulated secretion of the protein. Stimulation of AtT-20 cells with either forskolin or phorbol 12-myristate 13-acetate induces the secretion of wild-type CPE and of CPE lacking the pro region to similar extents, indicating a similar efficiency of sorting of the mutant. When the pro region of proalbumin is replaced with the pro region of CPE followed by expression in AtT-20 cells, the protein is not sorted into the regulated pathway, based on the lack of stimulated secretion. Confocal microscopy suggests that the proCPE/albumin protein is retained in the endoplasmic reticulum to a greater extent than is proalbumin. Pulse-chase analysis indicates that the pro region of CPE is not efficiently removed from the N-terminus of albumin, and the small amount of propeptide cleavage that does occur takes place soon before secretion of the protein. In contrast, confocal microscopy indicates that the majority of the propeptide is removed from CPE, and that this cleavage occurs in the trans-Golgi network or soon after sorting into the secretory vesicles. Taken together, these results suggest that the pro region of CPE is not required for the expression or intracellular routeing of this protein.

scholarly journals Dynamics of β-cell turnover: evidence for β-cell turnover and regeneration from sources of β-cells other than β-cell replication in the HIP rat

2009 ◽  
Vol 297 (2) ◽  
pp. E323-E330 ◽  
Author(s):  
Erica Manesso ◽  
Gianna M. Toffolo ◽  
Yoshifumi Saisho ◽  
Alexandra E. Butler ◽  
Aleksey V. Matveyenko ◽  
...  
Keyword(s):  
Cell Mass ◽  
Cell Formation ◽  
Cell Regeneration ◽  
Cell Turnover ◽  
Cell Replication ◽  
Wild Type ◽  
Β Cells ◽  
Β Cell ◽  
Islet Amyloid

Type 2 diabetes is characterized by hyperglycemia, a deficit in β-cells, increased β-cell apoptosis, and islet amyloid derived from islet amyloid polypeptide (IAPP). These characteristics are recapitulated in the human IAPP transgenic (HIP) rat. We developed a mathematical model to quantify β-cell turnover and applied it to nondiabetic wild type (WT) vs. HIP rats from age 2 days to 10 mo to establish 1) whether β-cell formation is derived exclusively from β-cell replication, or whether other sources of β-cells (OSB) are present, and 2) to what extent, if any, there is attempted β-cell regeneration in the HIP rat and if this is through β-cell replication or OSB. We conclude that formation and maintenance of adult β-cells depends largely (∼80%) on formation of β-cells independent from β-cell duplication. Moreover, this source adaptively increases in the HIP rat, implying attempted β-cell regeneration that substantially slows loss of β-cell mass.

scholarly journals ZnT8 Haploinsufficiency Impacts MIN6 Cell Zinc Content and β-Cell Phenotype via ZIP-ZnT8 Coregulation

2019 ◽  
Vol 20 (21) ◽  
pp. 5485 ◽  
Author(s):  
Rebecca Lawson ◽  
Wolfgang Maret ◽  
Christer Hogstrand
Keyword(s):  
Cell Survival ◽  
Cellular Proliferation ◽  
Zinc Content ◽  
Zinc Homeostasis ◽  
Cell Phenotype ◽  
Human Populations ◽  
Β Cells ◽  
Β Cell ◽  
Min6 Cell ◽  
Zip Family

The zinc transporter ZnT8 (SLC30A8) localises to insulin secretory granules of β-cells where it facilitates zinc uptake for insulin crystallisation. ZnT8 abundance has been linked to β-cell survival and functional phenotype. However, the consequences of ZnT8 haploinsufficiency for β-cell zinc trafficking and function remain unclear. Since investigations in human populations have shown SLC30A8 truncating polymorphisms to decrease the risk of developing Type 2 Diabetes, we hypothesised that ZnT8 haploinsufficiency would improve β-cell function and maintain the endocrine phenotype. We used CRISPR/Cas9 technology to generate ZnT8 haploinsufficient mouse MIN6 β-cells and showed that ZnT8 haploinsufficiency is associated with downregulation of mRNAs for Slc39a8 and Slc39a14, which encode for the zinc importers, Znt- and Irt-related proteins 8 (ZIP8) and 14 (ZIP14), and with lowered total cellular zinc content. ZnT8 haploinsufficiency disrupts expression of a distinct array of important β-cell markers, decreases cellular proliferation via mitogen-activated protein (MAP) kinase cascades and downregulates insulin gene expression. Thus, ZnT8 cooperates with zinc importers of the ZIP family to maintain β-cell zinc homeostasis. In contrast to the hypothesis, lowered ZnT8 expression reduces MIN6 cell survival by affecting zinc-dependent transcription factors that control the β-cell phenotype.

scholarly journals Critical roles for the TSC-mTOR pathway in β-cell function

2009 ◽  
Vol 297 (5) ◽  
pp. E1013-E1022 ◽  
Author(s):  
Hiroyuki Mori ◽  
Ken Inoki ◽  
Darren Opland ◽  
Heike Münzberg ◽  
Eneida C. Villanueva ◽  
...  
Keyword(s):  
Glycemic Control ◽  
Cell Function ◽  
Mammalian Target Of Rapamycin ◽  
Cell Number ◽  
Cell Phenotype ◽  
Insulin Production ◽  
Β Cells ◽  
Β Cell ◽  
The Face ◽  
Β Cell Function

TSC1 is a tumor suppressor that associates with TSC2 to inactivate Rheb, thereby inhibiting signaling by the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). mTORC1 stimulates cell growth by promoting anabolic cellular processes, such as translation, in response to growth factors and nutrient signals. To test roles for TSC1 and mTORC1 in β-cell function, we utilized Rip2/ Cre to generate mice lacking Tsc1 in pancreatic β-cells ( Rip-Tsc1cKO mice). Although obesity developed due to hypothalamic Tsc1 excision in older Rip-Tsc1cKO animals, young animals displayed a prominent gain-of-function β-cell phenotype prior to the onset of obesity. The young Rip-Tsc1cKO animals displayed improved glycemic control due to mTOR-mediated enhancement of β-cell size, mass, and insulin production but not determinants of β-cell number (proliferation and apoptosis), consistent with an important anabolic role for mTOR in β-cell function. Furthermore, mTOR mediated these effects in the face of impaired Akt signaling in β-cells. Thus, mTOR promulgates a dominant signal to promote β-cell/islet size and insulin production, and this pathway is crucial for β-cell function and glycemic control.

scholarly journals AKT1 Regulates Endoplasmic Reticulum Stress and Mediates the Adaptive Response of Pancreatic β Cells

2020 ◽  
Vol 40 (11) ◽  
Author(s):  
Zhechu Peng ◽  
Richa Aggarwal ◽  
Ni Zeng ◽  
Lina He ◽  
Eileen X. Stiles ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Adaptive Response ◽  
Metabolic Stress ◽  
Cell Phenotype ◽  
Β Cells ◽  
Β Cell ◽  
Pancreatic Β Cells ◽  
Α Subunit ◽  
Growth And Survival

ABSTRACT Isoforms of protein kinase B (also known as AKT) play important roles in mediating insulin and growth factor signals. Previous studies have suggested that the AKT2 isoform is critical for insulin-regulated glucose metabolism, while the role of the AKT1 isoform remains less clear. This study focuses on the effects of AKT1 on the adaptive response of pancreatic β cells. Using a mouse model with inducible β-cell-specific deletion of the Akt1 gene (βA1KO mice), we showed that AKT1 is involved in high-fat-diet (HFD)-induced growth and survival of β cells but is unnecessary for them to maintain a population in the absence of metabolic stress. When unchallenged, βA1KO mice presented the same metabolic profile and β-cell phenotype as the control mice with an intact Akt1 gene. When metabolic stress was induced by HFD, β cells in control mice with intact Akt1 proliferated as a compensatory mechanism for metabolic overload. Similar effects were not observed in βA1KO mice. We further demonstrated that AKT1 protein deficiency caused endoplasmic reticulum (ER) stress and potentiated β cells to undergo apoptosis. Our results revealed that AKT1 protein loss led to the induction of eukaryotic initiation factor 2 α subunit (eIF2α) signaling and ER stress markers under normal-chow-fed conditions, indicating chronic low-level ER stress. Together, these data established a role for AKT1 as a growth and survival factor for adaptive β-cell response and suggest that ER stress induction is responsible for this effect of AKT1.

scholarly journals Inefficient Secretion of Human H27A-Prolactin, a Mutant That Does Not Bind Zn2+

1997 ◽  
Vol 11 (10) ◽  
pp. 1544-1551 ◽  
Author(s):  
Zhenyu Sun ◽  
Min S. Lee ◽  
Harrison K. Rhee ◽  
Joanne M. Arrandale ◽  
Priscilla S. Dannies
Keyword(s):  
Secretory Pathway ◽  
Mutant Protein ◽  
Pituitary Cells ◽  
Wild Type ◽  
Rt Pcr ◽  
Selection Of

Abstract Human PRL binds Zn2+, but the function of the binding is not known. We investigated the effect on PRL production in pituitary cells by obtaining clones of GH4C1 cells stably transfected with human H27A-PRL, a mutant that does not bind Zn2+. Unexpectedly, clones transfected with the mutant human PRL made little rat PRL. Untransfected GH4C1 cells made between 0.5 to 10 μg rat PRL/105 cells in 24 h. Clones transfected with vector alone (four of four), wild type human PRL (six of six), or with human K69A-PRL (two of two) made amounts of rat PRL in the same range. Clones transfected with human H27A-PRL (five of five) made 0.003–0.1 μg rat PRL/105 cells in 24 h, and the production of rat PRL mRNA was reduced. Human H27A-PRL was not efficiently secreted; 20–40% newly synthesized H27A-PRL was degraded by 60 min, and there was usually a delay in release of newly synthesized H27A-PRL. Reduction of rat PRL production is not mediated through the PRL receptor, because no sequences for the receptor in GH4C1 cells were detected by RT-PCR. Proteins involved in folding, such as BiP, were not specifically elevated in the H27A-PRL clones. In transient transfections, in which cells have not undergone selection, we found no evidence for disulfide-bonded aggregates of the mutant protein. The results indicate that Zn2+ binding stabilizes PRL in the secretory pathway; the instablility of the mutant protein may trigger effects that suppress rat PRL production directly or that indirectly result in selection of clones with low rat PRL production.

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