Modern roles for an ancient system. Intracellular Complement in the regulation of β cell function

Research output: ThesisDoctoral Thesis (compilation)

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Abstract

Type 2 diabetes (T2D) is characterised by defective insulin exocytosis from the pancreatic β cells, accompanied by insulin resistance. Reduced β cells mass is often seen in T2D individuals, caused by enhanced β cells apoptosis. It is now understood that several components drive β cells dysfunction and apoptosis. These are glucose-and lipo-toxicity, ER stress, pro-inflammatory cytokines, dysfunctions in autophagy, and β cells dedifferentiation. Our studies revealed novel functions of intracellular complement components in β cells, presenting a new link between complement and diabetes development. We found that C3 is upregulated in pancreatic islets during T2D as a factor against β cells dysfunction caused by attenuated autophagy. In paper I, we revealed a high expression of C3 in human pancreatic islets. C3 was found intracellularly in isolated human pancreatic β cells. We verified the binding between C3 and ATG16L1 within the cytosol. C3 was required to maintain autophagy activity in β cells, as evidenced by the massive accumulation of LC3-II puncta, indicating that in the absence of C3 autophagosomes do not fuse with lysosomes. Autophagy protects the β cells from injuries caused by exposure to stressors, such as lipotoxicity. When exposing the C3-knockout INS-1 cells to β cells autophagy inducers (palmitate and IAPP), we observed significantly increased cell death caused by autophagy insufficiency. In paper II, we showed that silencing of CD59 expression in rat β cells significantly suppressed insulin secretion. Moreover, removing the membrane-bound CD59 did not affect insulin secretion, suggesting that intracellular CD59 is involved in this function. We found that the CD59 mutant, lacking the GPI-anchor, was present intracellularly in the β cell line. Non-GPI anchored CD59 interacts with SNARE protein: VAMP2 and rescues insulin secretion. We showed that the GPI-anchor, which is necessary for CD59 complement inhibitory function, is not necessary for its ability to mediate insulin secretion. Two other mutations: W40E and C64Y, rescued insulin secretion. Studies showed that these mutations result in a loss of CD59 complement inhibitory functions. Our data suggest that there are different structural requirements for separate functions of CD59, which are: MAC inhibition and insulin secretion. In papers III and IV, using RNA sequencing, we revealed the presence of two CD59 isoforms lacking the GPI anchoring domain (replaced with the unique C-terminal domains). We named these isoforms IRIS-1 and IRIS-2 (Isoforms Rescuing Insulin Secretion 1 and 2). Both isoforms exist in human and mouse pancreatic islets. They colocalize with insulin granules and interact with SNARE exocytotic machinery, allowing for insulin secretion. Induction of glucotoxicity in primary, healthy human islets led to a significant decrease in IRIS-1 protein-level expression. We found that expression of both IRIS-1 and IRIS-2 is markedly reduced in islets isolated from T2D patients compared to healthy controls, suggesting that hyperglycaemia may be one of the factors resulting in reduced IRIS-1 and IRIS-2 expression in T2D individuals. Next, an electropositive patch was found in the C-terminal region of IRIS-1, suggesting potential interaction with DNA. We found that IRIS-1 localizes in the nuclei of pancreatic β cells. We confirmed that the C-terminal domain of IRIS-1 is localising it to the nucleus. Since robust localisation of IRIS-1 in the nucleus is observed only in some nuclei, it can suggest that IRIS-1 is localising in the nucleus depending on the differentiation state of the cells or in a subset of cells with different functional relevance. We found that IRIS-1 expressing cells displayed significantly higher expression levels of Urocortin 3 and Pdx 1 (markers of mature β cells, which loss marks the beginning of β cells dedifferentiation), suggesting that IRIS-1 may be required for maintaining β cells identity and function.
Original languageEnglish
QualificationDoctor
Awarding Institution
  • Protein Chemistry, Malmö
Supervisors/Advisors
  • Blom, Anna, Supervisor
  • King, Ben, Assistant supervisor
  • Renström, Erik, Assistant supervisor
  • Snogerup-Linse, Sara, Assistant supervisor
Award date2022 Jun 17
Place of PublicationLund
Publisher
ISBN (Print)978-91-8021-255-7
Publication statusPublished - 2022

Bibliographical note

Defence details
Date: 2022-06-17
Time: 09:00
Place: Agardh föreläsningssal, CRC, Jan Waldenströms gata 35, Skånes Universitetssjukhus i Malmö
External reviewer(S)
Name: Mevorach, Dror
Title: Professor
Affiliation: Hadassah-Hebrew University, Jerusalem, Israel

Subject classification (UKÄ)

  • Cell and Molecular Biology

Free keywords

  • Complement system
  • Diabetes mellitus
  • Insulin secretion
  • CD59
  • C3
  • Isoforms
  • SNARE proteins

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