Localization of HS in islets of normal and STZ-treated diabetic
mice
In this study, we used two HS antibodies to explore the detailed
localization of HS in murine islets. The antibody reacts with 10E4
epitope of HS recognizes N-sulfated glucosamine residue of HS, while the
3G10 antibody reacts with a neo-epitope of HS, which is generated by
heparitinase I digestion on sections (David et al. , 1992). The
staining results of both 10E4 and 3G10 antibodies showed that HS was
highly expressed in normal islets (Fig. 1A and 1B). However, minor
differences were observed. The 10E4 antibody staining in control group
only exists in intra-islets (Fig. 1A). The 3G10 antibody staining showed
that HS was not only highly expressed in the islets, but also
distributed in a net shape in the extracellular matrix around the
pancreatic acinar cells (Fig. 1B). Double-fluorescence staining of HS
with four islet endocrine hormones showed that 10E4 mainly expresses in
insulin-positive beta cells but less in other types of islet endocrine
cells, including glucagon-positive alpha cells, somatostatin-positive
delta cells, and pancreatic polypeptide (PP)-positive PP cells (Fig.
1A), whereas 3G10 covers most islet endocrine cell types (Fig. 1B).
Obviously different expression patterns between 10E4 and 3G10 were found
in STZ-treated mice. As in normal islets, 10E4 was co-localized with
insulin, which is reduced with damage beta cells in STZ mice (Fig. 1A).
Using 3G10 antibody, the amount of intra-islet HS was clearly decreased
in the whole islets of STZ-treated mice, and HS exhibited a similar
pattern of net shape to that in the peripheral acinar cells (Fig. 1B).
It should be noticed that residual 3G10-stained HS could be still
observed in alpha cells, delta cells, and PP cells in STZ mice (Fig.
1B).
Heparanase is highly expressed in pancreatic beta cells and
monocytes in islets. Immunostaining showed that heparanase is expressed
in islets rather than peripheral acinar cells (Fig. 2A and 2B).
Remarkably, intra-islet content of heparanase is increased in
STZ-treated mice, in which we noticed some cells had extremely higher
expression of heparanase (Fig. 2A and 2B). We further analyzed the
expression level of heparanase in different islet cell types from
reported single cell sequencing data. Although the difference of
heparanase expression between diabetic and normal samples was not
significant due to limited sample number, it confirmed the expression of
heparanase is high in the alpha and beta endocrine cells in the islets
(Fig. 2C). To confirm this, double-immunofluorescence was used to
investigate the detailed localization of heparanase in islets. In
control mice, less F4/80-positive monocytes could be observed (Fig. 2D),
heparanase was expressed in most insulin-positive beta cells (Fig. 2E).
In contrast, heparanase was found to co-localized with F4/80-positive
monocytes in STZ islets, and heparanase-positive cells was still present
in islet without F4/80 (Fig. 2D). As shown in Fig. 2E, those
F4/80-negative cells that expressed heparanase was co-localized with
insulin.