A role for C3 in homeostatic autophagy
Besides targeting extracellularly opsonised bacteria for autophagy once
they enter the cell, C3 has been found to be important in maintaining
basal flux of autophagy in pancreatic β-cells. Autophagy is essential as
an adaptive response during development of insulin resistance in
peripheral tissues, a state of increased insulin demand that places
extra stress on insulin-producing β-cells. If β-cells are unable to keep
pace with the metabolic demand and die off, insulin production is halted
and overt type 2 diabetes develops. Autophagy acts against the breakdown
of pancreatic islet architecture and the failure of β-cells to sustain
sufficient insulin secretion, and therefore aids the maintenance of
glucose tolerance (Watada & Fujitani, 2015), and has been demonstrated
to protect β-cells against apoptosis induced by ER stress
(Bachar-Wikstrom et al., 2013) or lipotoxicity (Ebato et al., 2008;
Kong, Wu, Sun, & Zhou, 2017). Autophagy is also required for
homesotasis of pancreatic islets under normal conditions (Jung et al.,
2008). Identifying β-cell-intrinsic triggers of autophagy therefore has
considerable value for advancing strategies to limit β-cell loss during
disease. The complement system has recently been shown to have a number
of non-traditional roles in diabetes development (King & Blom, 2017).
Recently, we reported a protective effect of C3 against apoptosis of
β-cells, attributed to a role in maintenance of homeostatic autophagy
(King et al., 2019). We found high C3 expression in isolated human
pancreatic islets, that was significantly further upregulated in islets
from T2D donors, correlating with donor body mass index and glycated
haemaglobin levels, a clinical marker of diabetes. This islet-specific
C3 upregulation was also identified in several rodent models of
diabetes. Surprisingly, we found that as well as being secreted, C3 had
a cytosolic distribution in human islets and clonal β-cells. We
therefore probed for C3 interacting partners using protein microarrays
and in parallel with Sorbara et al (Sorbara et al., 2018), found
an interaction between C3 and ATG16L1. To investigate this further, we
explored the autophagy phenotype of CRISPR/Cas9-mediated C3 knockouts in
insulinoma INS-1 832/13 cells, a widely used β-cell model cell line
(Hohmeier et al., 2000). Studying the resulting phenotype of C3
knockouts revealed a dramatic arrest in the autophagy pathway. C3
knockouts displayed accumulation of LC3-II puncta as measured by
immunoblotting, as well as observed by confocal microscopy. These puncta
did not further accumulate in the presence of a lysosomal inhibitor,
indicating an inhibited turnover of LC3-positive autophagosomes, rather
than increased rate of autophagosome formation. Heterozygote knockout
clones exhibited an intermediate level of autophagy inhibition,
indicating a gene dose-dependent effect. An increased level of
autophagic substrate p62 in C3 knockout clones confirmed autophagic
dysfunction, and electron microscopy displayed an accumulation of
autophagosome-like structures. Pancreatic islets isolated from C3
knockout mice also demonstrated accumulated P62 and LC3-II levels,
compared to WT mouse islets.
Autophagy-depended targeting of insulin granules to lysosomes plays an
important role in protein quality control and insulin turnover.
Consistent with this, C3 knockout clones also had a significant increase
in numbers of insulin granules, that translated into increased
glucose-stimulated insulin secretion. However, it is possible that some
of this is secreted as biologically inactive proinsulin, since the ELISA
assay does not discriminate between these two forms. Circulating plasma
proinsulin levels are also increased in patients with type 2 diabetes
mellitus, consistent with reports of diabetes-induced islet autophagy
dysfunction (Ji et al., 2019; Masini et al., 2009).
Finally, the involvement of C3 in the cytoprotective function of
autophagy in stressed β-cells was also confirmed in the C3 knockout
β-cell clones. Exposure to free fatty acids, used to model in
vivo glucolipotoxicity, led to increased apoptosis in β-cell clones
lacking C3 expression. These same conditions increased both C3
expression and autophagic turnover in normal cells. Knockdown of ATG7,
essential for homeostatic autophagy, also increased apoptosis in WT
cells. Similarly, exposure to islet amyloid polypeptide, an
amyloidogenic peptide hormone that forms insoluble toxic deposits in the
pancreas of human diabetic patients (Jurgens et al., 2011), triggered
upregulation of C3 and autophagy, and caused increased apoptosis in
C3-knockout cells.
Having demonstrated a requirement for C3 for autophagic homeostasis in
β-cells, it is therefore possible that a lack of C3 could lead to an
increased loss of insulin secreting cells in the face of metabolic
challenge, as has been observed for β-cell-specific autophagy geneAtg7 -deficient mice (Ebato et al., 2008; Jung et al., 2008). On
the other hand, chronically upregulated C3 expression observed in type 2
diabetes subjects may lead to hyperactivation of autophagy and a literal
consumption of cellular insulin content. Further in vivoinvestigation of C3 involvement in maintaining insulin content and
β-cell mass is therefore required, including an assessment of
contributions of β-cell-derived C3, compared to serum-derived C3. The
hypothetical existence of separate pools of C3 within the cell, both
within the conventional secretory pathway and also within the cytosol,
also provides challenges to investigating these separately.