Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Insulin secretion and ASNA-1-dependent function of the endoplasmic reticulum in C. elegans
Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Surgery. (Peter Naredi)ORCID iD: 0000-0001-5824-6263
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

ASNA1 is a well-conserved ATPase involved in a wide range of functions, including cisplatin resistance, growth control, insulin secretion and targeting of tail-anchored (TA) proteins to membranes. It is a positive regulator of insulin secretion both in the roundworm Caenorhabditis elegans and in humans. Insulin secretion and downstream insulin/IGF signalling (IIS) stands at the heart of many human pathologies, such as diabetes, Alzheimer’s disease and cancer. A better understanding of IIS may therefore prove vital for treatment and cure of these diseases. This thesis aims to further investigate the function of asna-1, and to identify new regulators of IIS based on the asna-1 phenotype in C. elegans.

Worms lacking ASNA-1 arrest growth in the first larval stage, L1, with reduced insulin secretion. The L1 arrest represents the strongest of the IIS phenotypes in worms. Most regulators of the insulin pathway have been identified in screens for other IIS phenotypes, influencing lifespan or the dauer diapause. Therefore, new regulators could be found by screening for genes which, when inactivated, cause an asna-1-like L1 arrest. Using bioinformatic approaches, a set of 143 putative asna-1 interactors were identified, based on their predicted or confirmed interaction with asna-1 in various organisms. Depletion of the Golgi SNARE homologue YKT-6 or the mitochondrial translocase homologue TOMM-40 caused asna-1-like larval arrests. Using several criteria, including genetic suppression by daf-16/Foxo, it was established that YKT-6 and TOMM-40 are positive regulators of IIS. Both proteins were also required for normal DAF-28/insulin secretion.

Further investigation of TOMM-40 identified it as a ubiquitously expressed mitochondrial translocase in C. elegans: It localized to mitochondrial membranes and was required for importing a tagged mitochondrial reporter across mitochondrial membranes. Depletion of TOMM-40 caused a collapse of the proton gradient across the inner mitochondrial membrane and triggered the mitochondrial unfolded protein response (UPR). Worms with defective mitochondria failed to grow normally in presence of food, but this growth defect was suppressed by daf-16(mgDf50). In addition, tomm-40(RNAi) led to DAF-16/FOXO activation, an effect that was suppressed by over expression of DAF-28/insulin. Taken together, these findings support a model whereby signals of food availability are conveyed through respiring mitochondria to promote DAF-28/insulin secretion, which in turn promotes growth.

Biochemical studies have identified ASNA-1 as a chaperone that targets a subset of newly synthesized TA proteins to a receptor at the endoplasmic reticulum (ER) membrane. However, these findings have not been tested in vivo in a metazoan model. A reporter-based system to analyse TA protein targeting into the ER in live animals using confocal microscopy was set up. A model asna-1-dependent TA protein, Y38F2AR.9/SEC-61β, required functional ASNA-1 for correct targeting to the ER. Conversely, a model asna-1-independent TA protein, CYTB5.1/cytochrome B5, did not. This phenotype was shared with the predicted asna-1 receptor homologue, wrb-1. Consistently, WRB-1 was found to localize to the ER. However, other wrb-1 mutant phenotypes only partially overlap with those of asna-1 mutants, suggesting that ASNA-1 is either partially independent of WRB-1 for TA protein targeting or that ASNA-1 has additional functions besides its role in TA protein targeting.

Confocal microscopy also indicated that the ER morphology was aberrant in asna-1 and wrb-1 mutants. ER UPR was elevated in the asna-1 mutants, as indicated by the upregulation of an hsp-4/BiP reporter. Transmission and immuno-electron microscopy of these mutants revealed a swollen ER lumen, which is another hallmark of ER stress. High levels of autophagy in asna-1 animals and the presence of ER-containing autophagosomes in both asna-1 and wrb-1 mutants indicated a stress-induced remodelling of the ER membrane in these two mutants. In addition, both mutants had normal mitochondrial morphology, but showed severe effects on Golgi compartment morphology. Hypothetically, all these phenotypes could be due to defects in the signal recognition particle (SRP) pathway. This is because Y38F2AR.9/SEC-61β is both a TA protein and a component of the SEC-61 translocon. However, both Golgi and ER morphology was normal in Y38F2AR.9/sec-61β(tm1986) mutant animals, suggesting that the organellar defects seen in asna-1 and wrb-1 were due to a TA protein-dependent mechanism rather than an SRP-dependent mechanism. In addition, asna-1 mutants displayed numerous protein aggregates, consistent with a proposed role for ASNA-1 in shielding aggregation-prone TA protein membrane anchors from the hydrophilic environment of the cytosol.

In conclusion, YKT-6 and TOMM-40 are positive regulators of IIS and DAF-28/insulin secretion, implicating roles for Golgi and mitochondria in IIS. DAF-28 is a metabolically regulated insulin in C. elegans, since its secretion depends on active mitochondria. Mutants for asna-1 and its predicted receptor wrb-1 show severe defects in ER and Golgi morphology. These defects may occur because TA protein targeting in asna-1 and wrb-1 mutants is defective, which is also demonstrated here in the first analysis of this process in live animals.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet , 2014. , 80 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1634
National Category
Cell and Molecular Biology
Research subject
cellforskning; Genetics
Identifiers
URN: urn:nbn:se:umu:diva-85906ISBN: 978-91-7601-004-4 (print)OAI: oai:DiVA.org:umu-85906DiVA: diva2:696117
Public defence
2014-03-14, Hörsal Betula, by 6M, Norrlands Universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2014-02-21 Created: 2014-02-13 Last updated: 2014-02-21Bibliographically approved
List of papers
1. A directed RNAi screen based on larval growth arrest reveals new modifiers of C. elegans insulin signaling
Open this publication in new window or tab >>A directed RNAi screen based on larval growth arrest reveals new modifiers of C. elegans insulin signaling
Show others...
2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 4, e34507- p.Article in journal (Refereed) Published
Abstract [en]

Genes regulating Caenorhabditis elegans insulin/IGF signaling (IIS) have largely been identified on the basis of their involvement in dauer development or longevity. A third IIS phenotype is the first larval stage (L1) diapause, which is also influenced by asna-1, a regulator of DAF-28/insulin secretion. We reasoned that new regulators of IIS strength might be identified in screens based on the L1 diapause and the asna-1 phenotype. Eighty-six genes were selected for analysis by virtue of their predicted interaction with ASNA-1 and screened for asna-1-like larval arrest. ykt-6, mrps-2, mrps-10 and mrpl-43 were identified as genes which, when inactivated, caused larval arrest without any associated feeding defects. Several tests indicated that IIS strength was weaker and that insulin secretion was defective in these animals. This study highlights the role of the Golgi network and the mitochondria in insulin secretion and provides a new list of genes that modulate IIS in C. elegans.

National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:umu:diva-57394 (URN)10.1371/journal.pone.0034507 (DOI)000305338600033 ()
Available from: 2012-07-17 Created: 2012-07-16 Last updated: 2017-12-07Bibliographically approved
2. Mitochondrial function is required for secretion of DAF-28/insulin in C. elegans.
Open this publication in new window or tab >>Mitochondrial function is required for secretion of DAF-28/insulin in C. elegans.
2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 1, e14507- p.Article in journal (Refereed) Published
Abstract [en]

While insulin signaling has been extensively studied in Caenorhabditis elegans in the context of ageing and stress response, less is known about the factors underlying the secretion of insulin ligands upstream of the insulin receptor. Activation of the receptor governs the decision whether to progress through the reproductive lifecycle or to arrest growth and enter hibernation. We find that animals with reduced levels of the mitochondrial outer membrane translocase homologue TOMM-40 arrest growth as larvae and have decreased insulin signaling strength. TOMM-40 acts as a mitochondrial translocase in C. elegans and in its absence animals fail to import a mitochondrial protein reporter across the mitochondrial membrane(s). Inactivation of TOMM-40 evokes the mitochondrial unfolded protein response and causes a collapse of the proton gradient across the inner mitochondrial membrane. Consequently these broadly dysfunctional mitochondria render an inability to couple food abundance to secretion of DAF-28/insulin. The secretion defect is not general in nature since two other neuropeptides, ANF::GFP and INS-22::VENUS, are secreted normally. RNAi against two other putative members of the TOMM complex give similar phenotypes, implying that DAF-28 secretion is sensitive to mitochondrial dysfunction in general. We conclude that mitochondrial function is required for C. elegans to secrete DAF-28/insulin when food is abundant. This modulation of secretion likely represents an additional level of control over DAF-28/insulin function.

Place, publisher, year, edition, pages
Public Library of Science, 2011
National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:umu:diva-41185 (URN)10.1371/journal.pone.0014507 (DOI)21264209 (PubMedID)
Available from: 2011-03-21 Created: 2011-03-18 Last updated: 2017-12-11Bibliographically approved
3. ASNA-1 acts independently of its endoplasmic reticulum receptor WRB-1 to promote insulin/IGF signalling
Open this publication in new window or tab >>ASNA-1 acts independently of its endoplasmic reticulum receptor WRB-1 to promote insulin/IGF signalling
(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Research subject
Genetics; cellforskning
Identifiers
urn:nbn:se:umu:diva-85905 (URN)
Available from: 2014-02-13 Created: 2014-02-13 Last updated: 2014-02-24Bibliographically approved

Open Access in DiVA

fulltext(31836 kB)1029 downloads
File information
File name FULLTEXT02.pdfFile size 31836 kBChecksum SHA-512
b17fb992de23682bd4e8a79908725bbc5c062771ee1786f73ff1cbb580f209672d732be4206dc44cadc36b1246c25b0b627aea95f2dca74fa175c3639538f267
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Billing, Ola
By organisation
Surgery
Cell and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 1029 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 635 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf