scholarly journals A Novel Nonsense INS Mutation Causes Inefficient Preproinsulin Translocation Into the Endoplasmic Reticulum

2022 ◽  
Vol 12 ◽  
Author(s):  
Ying Yang ◽  
Hua Shu ◽  
Jingxin Hu ◽  
Lei Li ◽  
Jianyu Wang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Neonatal Diabetes ◽  
Early Termination ◽  
Insulin Biosynthesis ◽  
Regulation Mechanism ◽  
Metabolic Labeling ◽  
Insulin Production ◽  
Wild Type ◽  
Β Cell ◽  
Whole Exome

Preproinsulin (PPI) translocation across the membrane of the endoplasmic reticulum (ER) is the first and critical step of insulin biosynthesis. Inefficient PPI translocation caused by signal peptide (SP) mutations can lead to β-cell failure and diabetes. However, the effect of proinsulin domain on the efficiency of PPI translocation remains unknown. With whole exome sequencing, we identified a novel INS nonsense mutation resulting in an early termination at the 46th residue of PPI (PPI-R46X) in two unrelated patients with early-onset diabetes. We examined biological behaviors of the mutant and compared them to that of an established neonatal diabetes causing mutant PPI-C96Y. Although both mutants were retained in the cells, unlike C96Y, R46X did not induce ER stress or form abnormal disulfide-linked proinsulin complexes. More importantly, R46X did not interact with co-expressed wild-type (WT) proinsulin in the ER, and did not impair proinsulin-WT folding, trafficking, and insulin production. Metabolic labeling experiments established that, despite with an intact SP, R46X failed to be efficiently translocated into the ER, suggesting that proinsulin domain downstream of SP plays an important unrecognized role in PPI translocation across the ER membrane. The study not only expends the list of INS mutations associated with diabetes, but also provides genetic and biological evidence underlying the regulation mechanism of PPI translocation.

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

2016 ◽  
Vol 23 (1) ◽  
pp. 179-193 ◽  
Author(s):  
Marta Szabat ◽  
Melissa M. Page ◽  
Evgeniy Panzhinskiy ◽  
Søs Skovsø ◽  
Majid Mojibian ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
Insulin Production ◽  
Β Cell

scholarly journals Impact of scavenging hydrogen peroxide in the endoplasmic reticulum for β cell function

2015 ◽  
Vol 55 (1) ◽  
pp. 21-29 ◽  
Author(s):  
S Lortz ◽  
S Lenzen ◽  
I Mehmeti
Keyword(s):  
Gene Expression ◽  
Hydrogen Peroxide ◽  
Endoplasmic Reticulum ◽  
Insulin Secretion ◽  
Er Stress ◽  
Insulin Gene ◽  
Insulin Content ◽  
Insulin Biosynthesis ◽  
Β Cell ◽  
Insulin Gene Expression

Oxidative folding of nascent proteins in the endoplasmic reticulum (ER), catalysed by one or more members of the protein disulfide isomerase family and the sulfhydryl oxidase ER oxidoreductin 1 (ERO1), is accompanied by generation of hydrogen peroxide (H2O2). Because of the high rate of insulin biosynthesis and the low expression of H2O2-inactivating enzymes in pancreatic β cells, it has been proposed that the luminal H2O2concentration might be very high. As the role of this H2O2in ER stress and proinsulin processing is still unsolved, an ER-targeted and luminal-active catalase variant, ER-Catalase N244, was expressed in insulin-secreting INS-1E cells. In these cells, the influence of ER-specific H2O2removal on cytokine-mediated cytotoxicity and ER stress, insulin gene expression, insulin content and secretion was analysed. The expression of ER-Catalase N244 reduced the toxicity of exogenously added H2O2significantly with a threefold increase of the EC50value for H2O2. However, the expression of cytokine-induced ER stress genes and viability after incubation with β cell toxic cytokines (IL1β alone or together with TNFα+IFNγ) was not affected by ER-Catalase N244. In control and ER-Catalase N244 expressing cells, insulin secretion and proinsulin content was identical, while removal of luminal H2O2reduced insulin gene expression and insulin content in ER-Catalase N244 expressing cells. These data show that ER-Catalase N244 reduced H2O2toxicity but did not provide protection against pro-inflammatory cytokine-mediated toxicity and ER stress. Insulin secretion was not affected by decreasing H2O2in the ER in spite of a reduced insulin transcription and processing.

scholarly journals Oxidative and endoplasmic reticulum stress in β-cell dysfunction in diabetes

2015 ◽  
Vol 56 (2) ◽  
pp. R33-R54 ◽  
Author(s):  
Sumaira Z Hasnain ◽  
Johannes B Prins ◽  
Michael A McGuckin
Keyword(s):  
Oxidative Stress ◽  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Inflammatory Cytokines ◽  
Cell Stress ◽  
Insulin Biosynthesis ◽  
Central Feature ◽  
Β Cells ◽  
Β Cell ◽  
Cell Dysfunction

The inability of pancreatic β-cells to make sufficient insulin to control blood sugar is a central feature of the aetiology of most forms of diabetes. In this review we focus on the deleterious effects of oxidative stress and endoplasmic reticulum (ER) stress on β-cell insulin biosynthesis and secretion and on inflammatory signalling and apoptosis with a particular emphasis on type 2 diabetes (T2D). We argue that oxidative stress and ER stress are closely entwined phenomena fundamentally involved in β-cell dysfunction by direct effects on insulin biosynthesis and due to consequences of the ER stress-induced unfolded protein response. We summarise evidence that, although these phenomenon can be driven by intrinsic β-cell defects in rare forms of diabetes, in T2D β-cell stress is driven by a range of local environmental factors including increased drivers of insulin biosynthesis, glucolipotoxicity and inflammatory cytokines. We describe our recent findings that a range of inflammatory cytokines contribute to β-cell stress in diabetes and our discovery that interleukin 22 protects β-cells from oxidative stress regardless of the environmental triggers and can correct much of diabetes pathophysiology in animal models. Finally we summarise evidence that β-cell dysfunction is reversible in T2D and discuss therapeutic opportunities for relieving oxidative and ER stress and restoring glycaemic control.

scholarly journals Role of malectin in Glc2Man9GlcNAc2-dependent quality control of α1-antitrypsin

2011 ◽  
Vol 22 (19) ◽  
pp. 3559-3570 ◽  
Author(s):  
Yang Chen ◽  
Dan Hu ◽  
Rikio Yabe ◽  
Hiroaki Tateno ◽  
Sheng-Ying Qin ◽  
...  
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Structural Similarity ◽  
Metabolic Labeling ◽  
Wild Type ◽  
Binding Assays ◽  
Mrna Transcription ◽  
Glycoprotein Quality Control

Malectin was first discovered as a novel endoplasmic reticulum (ER)–resident lectin from Xenopus laevis that exhibits structural similarity to bacterial glycosylhydrolases. Like other intracellular lectins involved in glycoprotein quality control, malectin is highly conserved in animals. Here results from in vitro membrane-based binding assays and frontal affinity chromatography confirm that human malectin binds specifically to Glc2Man9GlcNAc2 (G2M9) N-glycan, with a Ka of 1.97 × 105 M−1, whereas binding to Glc1Man9GlcNAc2 (G1M9), Glc3Man9GlcNAc2 (G3M9), and other N-glycans is barely detectable. Metabolic labeling and immunoprecipitation experiments demonstrate that before entering the calnexin cycle, the folding-defective human α1-antitrypsin variant null Hong Kong (ATNHK) stably associates with malectin, whereas wild-type α1-antitrypsin (AT) or N-glycan–truncated variant of ATNHK (ATNHK-Q3) dose not. Moreover, malectin overexpression dramatically inhibits the secretion of ATNHK through a mechanism that involves enhanced ER-associated protein degradation; by comparison, the secretion of AT and ATNHK-Q3 is only slightly affected by malectin overexpression. ER-stress induced by tunicamycin results in significantly elevated mRNA transcription of malectin. These observations suggest a possible role of malectin in regulating newly synthesized glycoproteins via G2M9 recognition.

scholarly journals Ste6p Mutants Defective in Exit from the Endoplasmic Reticulum (ER) Reveal Aspects of an ER Quality Control Pathway inSaccharomyces cerevisiae

1998 ◽  
Vol 9 (10) ◽  
pp. 2767-2784 ◽  
Author(s):  
Diego Loayza ◽  
Amy Tam ◽  
Walter K. Schmidt ◽  
Susan Michaelis
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Subcellular Fractionation ◽  
Metabolic Labeling ◽  
Wild Type ◽  
Er Quality Control ◽  
Ubiquitin Proteasome ◽  
Mutant Forms ◽  
Er Membrane

We are studying the intracellular trafficking of the multispanning membrane protein Ste6p, the a-factor transporter inSaccharomyces cerevisiae and a member of the ATP-binding cassette superfamily of proteins. In the present study, we have used Ste6p as model for studying the process of endoplasmic reticulum (ER) quality control, about which relatively little is known in yeast. We have identified three mutant forms of Ste6p that are aberrantly ER retained, as determined by immunofluorescence and subcellular fractionation. By pulse-chase metabolic labeling, we demonstrate that these mutants define two distinct classes. The single member of Class I, Ste6–166p, is highly unstable. We show that its degradation involves the ubiquitin–proteasome system, as indicated by its in vivo stabilization in certain ubiquitin–proteasome mutants or when cells are treated with the proteasome inhibitor drug MG132. The two Class II mutant proteins, Ste6–13p and Ste6–90p, are hyperstable relative to wild-type Ste6p and accumulate in the ER membrane. This represents the first report of a single protein in yeast for which distinct mutant forms can be channeled to different outcomes by the ER quality control system. We propose that these two classes of ER-retained Ste6p mutants may define distinct checkpoint steps in a linear pathway of ER quality control in yeast. In addition, a screen for high-copy suppressors of the mating defect of one of the ER-retained ste6 mutants has identified a proteasome subunit, Hrd2p/p97, previously implicated in the regulated degradation of wild-type hydroxymethylglutaryl-CoA reductase in the ER membrane.

scholarly journals The Carboxyl-Terminal Region of Protein C Is Essential for Its Secretion

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3784-3791 ◽  
Author(s):  
Akira Katsumi ◽  
Tetsuhito Kojima ◽  
Takao Senda ◽  
Tomio Yamazaki ◽  
Hiroaki Tsukamoto ◽  
...  
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Protein C ◽  
Terminal Region ◽  
Metabolic Labeling ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein ◽  
Guanine Residue ◽  
Carboxyl Terminal

We have previously reported a mutated protein C, designated protein C Nagoya (PCN), characterized by the deletion of a single guanine residue (8857G). This frameshift mutation results in the replacement of the carboxyl-terminal 39 amino acids of wild-type protein C (G381-P419) by 81 abnormal amino acids. This elongated mutant was not effectively secreted, and was retained in the endoplasmic reticulum. To determine why PCN is not secreted, we constructed a series of mutants from which some or all of the 81 amino acids were deleted. None of these shortened proteins were secreted from producing cells, indicating that the carboxyl-terminal extension is not mainly responsible for the intracellular retention of PCN, and that the 39 carboxyl-terminal amino acids of wild-type protein C are required for secretion. To determine which residues are essential for the secretion of protein C, deletion mutants of the carboxyl-terminal region (D401-P419) were prepared. Metabolic labeling showed that mutants of protein C truncated before W417, Q414, E411, or K410 were efficiently secreted. On the other hand, the mutants truncated before D409 were retained and degraded intracellularly. Immunofluorescence and immunoelectron microscopy showed that truncation before D409 blocks the movement from rough endoplasmic reticulum to the Golgi apparatus. To understand the conformational change in the carboxyl-terminal region, two models of truncated activated protein C were constructed using energy optimization and molecular dynamics with water molecules.

scholarly journals The Carboxyl-Terminal Region of Protein C Is Essential for Its Secretion

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3784-3791 ◽  
Author(s):  
Akira Katsumi ◽  
Tetsuhito Kojima ◽  
Takao Senda ◽  
Tomio Yamazaki ◽  
Hiroaki Tsukamoto ◽  
...  
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Protein C ◽  
Terminal Region ◽  
Metabolic Labeling ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein ◽  
Guanine Residue ◽  
Carboxyl Terminal

Abstract We have previously reported a mutated protein C, designated protein C Nagoya (PCN), characterized by the deletion of a single guanine residue (8857G). This frameshift mutation results in the replacement of the carboxyl-terminal 39 amino acids of wild-type protein C (G381-P419) by 81 abnormal amino acids. This elongated mutant was not effectively secreted, and was retained in the endoplasmic reticulum. To determine why PCN is not secreted, we constructed a series of mutants from which some or all of the 81 amino acids were deleted. None of these shortened proteins were secreted from producing cells, indicating that the carboxyl-terminal extension is not mainly responsible for the intracellular retention of PCN, and that the 39 carboxyl-terminal amino acids of wild-type protein C are required for secretion. To determine which residues are essential for the secretion of protein C, deletion mutants of the carboxyl-terminal region (D401-P419) were prepared. Metabolic labeling showed that mutants of protein C truncated before W417, Q414, E411, or K410 were efficiently secreted. On the other hand, the mutants truncated before D409 were retained and degraded intracellularly. Immunofluorescence and immunoelectron microscopy showed that truncation before D409 blocks the movement from rough endoplasmic reticulum to the Golgi apparatus. To understand the conformational change in the carboxyl-terminal region, two models of truncated activated protein C were constructed using energy optimization and molecular dynamics with water molecules.

scholarly journals Antipsychotic olanzapine-induced misfolding of proinsulin in the endoplasmic reticulum accounts for atypical development of diabetes

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Satoshi Ninagawa ◽  
Seiichiro Tada ◽  
Masaki Okumura ◽  
Kenta Inoguchi ◽  
Misaki Kinoshita ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Bond Formation ◽  
Insulin Biosynthesis ◽  
Β Cell ◽  
Cell Dysfunction ◽  
Subsequent Decrease ◽  
Insulin Granules ◽  
Second Generation Antipsychotics ◽  
Atypical Development ◽  
Primary Localization

Second-generation antipsychotics are widely used to medicate patients with schizophrenia, but may cause metabolic side effects such as diabetes, which has been considered to result from obesity-associated insulin resistance. Olanzapine is particularly well known for this effect. However, clinical studies have suggested that olanzapine-induced hyperglycemia in certain patients cannot be explained by such a generalized mechanism. Here, we focused on the effects of olanzapine on insulin biosynthesis and secretion by mouse insulinoma MIN6 cells. Olanzapine reduced maturation of proinsulin, and thereby inhibited secretion of insulin; and specifically shifted the primary localization of proinsulin from insulin granules to the endoplasmic reticulum. This was due to olanzapine’s impairment of proper disulfide bond formation in proinsulin, although direct targets of olanzapine remain undetermined. Olanzapine-induced proinsulin misfolding and subsequent decrease also occurred at the mouse level. This mechanism of olanzapine-induced β-cell dysfunction should be considered, together with weight gain, when patients are administered olanzapine.

scholarly journals Jiedu Tongluo Tiaogan Formula Protects Pancreatic Islet Cells Against Dysfunction by Relieving Endoplasmic Reticulum Stress and Excessive Autophagy via Regulating CaMKKβ/AMPK Pathway

2021 ◽  
Author(s):  
Chunli Piao ◽  
Qi Zhang ◽  
Wenqi Jin ◽  
Han Wang ◽  
Cheng Tang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Pancreatic Injury ◽  
Insulin Biosynthesis ◽  
Β Cells ◽  
Β Cell ◽  
Cell Dysfunction ◽  
Ampk Pathway ◽  
Glucose Stimulated Insulin Secretion

Abstract Background: Endoplasmic reticulum stress (ERS) and excessive autophagy are increasingly recognized as risk factors associated with development and progression of β-cell dysfunction. Jiedu Tongluo Tiaogan Formula (JTTF) has known anti-glucotoxicity activities, but its pharmacological effects on pancreatic cell are not clearly understood. This study was designed to investigate JTTF effects/mechanisms on in vitro glucotoxicity (HG)-induced ERS and excessive autophagic damage of pancreatic cells in vitro and on in vivo pancreatic injury in db/db mice. Methods: The chemical composition of a JTTF preparation were analyzed using high-performance liquid chromatographic fingerprinting. Meanwhile, cell viability, glucose-stimulated insulin secretion, insulin biosynthesis dysfunction, Ca2+ overload, ERS and excessive autophagy were assessed in JTTF-pretreated pancreatic β-cells with HG-induced injury. Hematoxylin and eosin staining and immunohistochemical analyses of pancreatic tissues revealed effects of in vivo JTTF pretreatment on development of HG-induced pancreatic injury in db/db mice. Results: Five JTTF chemical components were identified. Our results revealed that JTTF treatment protected β-cells from HG injury by increasing insulin biosynthesis and glucose-stimulated insulin secretion (GSIS), while also decreasing Ca2+ overload, ERS and excessive autophagy. Furthermore, protective effects of JTTF treatment against HG-induced β-cell ERS and excessive autophagy were linked to regulation of CaMKKβ/AMPK pathway functions, while JTTF administration as confirmed to reverse pancreatic injury in db/db mice. Conclusions: Collectively, the results presented here indicate that JTTF may prevent islet cell dysfunction in T2DM mice by inhibiting CaMKKβ/AMPK pathway-mediated ERS and excessive autophagy. These findings enhance our understanding of mechanisms underlying beneficial JTTF-induced amelioration of T2DM.

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